Novel anti-TPBG antibodies and antibody-drug conjugates based thereon, their therapeutic methods and uses

Abstract

The present invention relates to a novel anti-TPBG antibody containing Fc silencing mutations such as leucine (L) to alanine (A) substitutions (LALA mutations) at positions 234 and 235, an antibody-drug conjugate (ADC) based thereon, and therapeutic methods and their use, particularly relating to cancer treatment.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to European Patent Application No. 22202240, filed with the European Patent Office on 18 October 2022, which for all purposes is incorporated herein by reference in its entirety.

[0002] This application claims priority to European Patent Application No. 23172971, filed with the European Patent Office on 11 May 2023, which for all purposes is incorporated herein by its entirety.

[0003] Sequence List This application includes a sequence listing in a computer-readable form, which is incorporated herein by reference.

[0004] Technical field of inventions The present invention relates to a novel anti-TPBG antibody containing Fc silencing mutations such as leucine (L) to alanine (A) substitutions (LALA mutations) at positions 234 and 235, a novel antibody-drug conjugate (ADC) based thereon, and therapeutic methods and their use, particularly for cancer treatment. [Background technology]

[0005] Background of the Invention Carcinoembryonic antigen 5T4 (synonymously also known as trophoblast glycoprotein (TPBG)) is a 72kDa glycoprotein typically expressed only during embryonic development, with very limited expression in normal mature tissues. However, 5T4 expression has been reported to be significantly upregulated in many types of carcinoma, including non-limited lung, breast, gastric, prostate, colon, and ovarian cancers, and its expression has been correlated with poor prognosis in several target diseases. Given these characteristics, 5T4 is considered a critical cancer target, and various therapeutic approaches targeting 5T4, including antibody-drug conjugates, bispecific T-cell engagers, CAR-T approaches, and cancer vaccines, are being investigated. Humanized mAb H8 is known in the art (e.g., Shaw et al., 2000, Biochim Biophys Acta 2000 Dec 15;1524(2-3):238-46) and was derived from the original mouse 5T4 monoclonal antibody that binds to TPBG (Hole and Stern, 1988 and Shaw et al., 2000). Several first-generation antibody-drug conjugates have been developed in the past, but these have been discontinued, partly due to toxicity related to the linker-payload.

[0006] Therefore, there is a need for novel antibodies with improved characteristics, and ADCs based on them that have improved toxicity profiles and higher functionality. Accordingly, the technical objective of the present invention is to satisfy this need.

[0007] The present invention satisfies these needs by providing a novel anti-TPBG antibody containing Fc silencing mutations, such as leucine (L) to alanine (A) substitutions (LALA mutations) at positions 234 and 235 in the Fc region, which are capable of reducing the effector function of immune cells; a novel TPBG-targeted antibody-drug conjugate (ADC) based thereon (which is produced by applying P5 conjugation technology (e.g., WO2018 / 041985), which is based on modification of interchain cysteine ​​residues with an unsaturated phosphoneamidate reagent); and therapeutic methods and their use, particularly relating to cancer treatment. [Overview of the project]

[0008] This technical challenge is resolved by the provisions defined in the claims.

[0009] Therefore, the present invention relates to an anti-TPBG antibody (e.g., an antibody against TPBG_human trophoblast glycoprotein (e.g., having UniProt accession number: Q13641 or SEQ ID NO: 1)), wherein the anti-TPBG antibody comprises an Fc silencing mutation such as a leucine (L) to alanine (A) substitution at positions 234 and 235 (LALA mutation), and the anti-TPBG antibody is capable of binding to human trophoblast glycoprotein (TPBG) (e.g., having UniProt accession number: Q13641 or SEQ ID NO: 1); and the anti-TPBG antibody is capable of binding to marmoset (e.g., common marmoset (Callithrix jacchus)) TPBG (e.g., UniProtKB accession number: F7I0T3 or SEQ ID NO: 1) with white tufts of hair around the ears. The present invention relates to an anti-TPBG antibody that is capable of cross-reactivity with (having 2) and is capable of internal transfer, preferably by antigen-mediated internal transfer of the antibody.

[0010] The present invention further relates to monoclonal human or humanized IgG1 anti-TPBG antibodies, preferably containing a kappa (κ) light chain.

[0011] The present invention also relates to a hybridoma that produces the antibody of the present invention.

[0012] The present invention also relates to nucleic acids encoding the antibodies of the present invention.

[0013] The present invention also relates to an expression vector comprising at least one of the nucleic acid molecules of the present invention.

[0014] The present invention also relates to isolated host cells (e.g., isolated recombinant host cells) containing the vector and / or nucleic acids of the present invention.

[0015] The present invention also relates to an antibody-drug conjugate (ADC) comprising the anti-TPBG antibody of the present invention.

[0016] The present invention also relates to antibody-drug conjugates (ADCs) of the present invention, comprising an anti-TPBG antibody of the present invention (e.g., a humanized monoclonal TPBG-specific IgG1 antibody) conjugated to a cytotoxic payload / drug; wherein (a) the cytotoxic payload is selected from the group consisting of camptothecin, mytansinoids, calicheamycin, duochamycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof; and / or (b) the cytotoxic payload is exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, vero (c) The cytotoxic payload is a camptothecin moiety C selected from the group consisting of tecan, lulutotecan, rubitecan, silatecan, cositecan, and gimatecan; and / or (c) the cytotoxic payload is conjugated via a cleavable linker (L), preferably the linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction, and more preferably the linker is cleavable by a protease, preferably a cathepsin such as cathepsin B; and / or (d) the linker (L) comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) the cytotoxic payload is exatecan conjugated via a chemical valine-citrulline-PAB free unit or a valine-alanine-PAB free unit, the free unit being cleavable by a protease.Preferably, the drug-to-antibody ratio (DAR) is in the range of 0 to 20, preferably 1 to 20, more preferably 2 to 10, even more preferably 4 to 8, and most preferably 4 or 8.

[0017] The present invention also relates to compositions or kits comprising the anti-TPBG, antibody-drug conjugate (ADC), hybridoma, nucleic acid, expression vector, and / or host cell of the present invention.

[0018] The present invention also relates to a method for synthesizing antibody-drug conjugates (ADCs) of the present invention.

[0019] The present invention further relates to methods of treatment, as well as to the use of the antibodies, antibody-drug conjugates (ADCs), nucleic acids, expression vectors, host cells, compositions, and / or kits of the present invention.

[0020] Sequence List Overview As described herein, unless otherwise specified, the UniProtKB accession number (https: / / www.uniprot.org / release-notes / 2022-08-03-release, for example, available in UniProt release 2022_03 published on August 3, 2022) is referred to.

[0021] This application includes a sequence listing in a computer-readable form, which is incorporated herein by reference. [Brief explanation of the drawing]

[0022] The present invention will be better understood by referring to the detailed description and considering in conjunction with the non-limiting embodiments and accompanying drawings, respectively. The drawings are shown below.

[0023] [Figure 1] The binding of the antibody of the present invention to human 5T4 cells, as evaluated by flow cytometry, is shown. The graph shows the mean value (n=2) ± SEM. [Figure 2] The binding of the antibody of the present invention to human 5T4, as evaluated by ELISA, is shown. The graph shows the mean (n=2) ± SEM. [Figure 3] The graph shows the binding of the antibody of the present invention to 5T4 marmosets with white tufts of hair around the ears, as evaluated by flow cytometry. The graph shows the mean (n=2) ± SD. [Figure 4] This shows the internal migration of the antibody of the present invention, as evaluated by flow cytometry. [Figure 5] It exhibits in vitro cytotoxicity in the target cell line, as evaluated by the resazurin assay. [Figure 6] This demonstrates the bystander activity of ADC. [Figure 7] This study demonstrates in vitro inhibition of topoisomerase I by exatecan delivery via ADC. [Figure 8] This study demonstrates the in vivo efficacy of ADCs in reducing tumor volume in a mouse xenograft model. [Figure 9] This study demonstrates the tolerability of ADC in relation to body weight in a mouse xenograft model. [Figure 10] This shows ADC's in vivo penalty kick evaluation. [Figure 11] The UV chromatogram obtained by LC / MS measurement of P5(PEG12)-COOH is shown. [Figure 12] The UV chromatogram obtained by LC / MS measurement of P5(PEG24)-OSu is shown. [Figure 13] UV chromatogram obtained by LC / MS measurement of NH2-VC-PAB-exatecan TFA salt. [Figure 14] UV chromatogram obtained by LC / MS measurement of NH2-VA-PAB-exatecan TFA salt. [Figure 15]UV chromatogram of P5(PEG2)-VC-PAB-exatecan by LC / MS measurement. [Figure 16] UV chromatogram of P5(PEG12)-VC-PAB-exatecan by LC / MS measurement. [Figure 17] UV chromatogram of P5(PEG24)-VC-PAB-exatecan by LC / MS measurement. [Figure 18] UV chromatogram of P5(PEG12)-VA-PAB-exatecan by LC / MS measurement. [Figure 19] UV chromatogram of P5(PEG12)-exatecan by LC / MS measurement. [Figure 20] Analytical characterization of an ADC conjugated to P5(PEG24)-VC-PAB-exatecan and purified by CEX from anti-TPBG antibody is shown. A) SEC for analysis, B) HIC for analysis, C) MS spectrum for analysis after conjugation. The data clearly show that the ADC is fully conjugated up to DAR8 and there are only a small amount of aggregates after purification. [Figure 21-1] Exemplary sequences of the anti-TPBG antibody of the present invention are shown. [Figure 21-2] Refer to the description of Figure 21-1. [Figure 22] Results of ex vivo serum stability analysis of anti-TPBG-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, are shown. [Figure 23-1] Results of in vivo efficacy analysis of anti-TPBG-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, in a patient-derived xenograft model (PDX) are shown. [Figure 23-2] Refer to the description of Figure 23-1. [Figure 23-3] Refer to the description of Figure 23-1. [Figure 23-4] Refer to the description of Figure 23-1. [Figure 24]Results of in vivo toxicity analysis of anti-TPBG-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, in marmoset monkeys are shown. [Figure 25] MS analysis of anti-TPBG comparative ADC1 synthesized by the method described herein is shown. Measured mass (in daltons) and absolute intensity are annotated to the signals. The drug-to-antibody ratio was estimated to be 4.2 from the MS signals. LC: light chain of anti-TPBG comparative antibody 1, HC: heavy chain of anti-TPBG comparative antibody 1. [Figure 26] MS analysis of anti-TPBG comparative ADC2 synthesized by the method described above is shown. Measured mass (in daltons) and absolute intensity are annotated to the signals. The drug-to-antibody ratio was estimated to be 1.7 from the MS signals. LC: light chain of anti-TPBG comparative antibody 2, HC: heavy chain of anti-TPBG comparative antibody 2. [Figure 27] Cytotoxicity dose responses of anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR 8 and corresponding non-targeting isotype control conjugates compared to anti-TPBG comparative ADC1 in four different cell lines are shown. Mean values and SD of two measurements, as well as dose-response fitting, are shown. Fluorescence (in %) compared to medium control is equal to the ratio (%) of live cells. [Figure 28] Cytotoxicity dose responses of anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR 8 and corresponding non-targeting isotype control conjugates compared to anti-TPBG comparative ADC2 in three different cell lines are shown. Mean values and SD of two measurements, as well as dose-response fitting, are shown. Fluorescence (in %) compared to medium control is equal to the ratio (%) of live cells. [Figure 29]This report shows the cytotoxic dose-response of target-negative cells (SW-620) after transferring cell culture supernatants from two different 5T4-positive cell lines (MDA-MB-468 and BXPC-3) pretreated with serial dilutions of anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR 8 or anti-TPBG comparative ADC2. The mean and standard deviation of the two measurements, as well as the dose-response fitting, are shown. Fluorescence (in %) compared to the medium control is equal to the percentage of viable cells (%). [Figure 30] This report shows the cytotoxic dose-response of serial dilutions of anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR 8 ADC in co-cultures of target-positive cell line (BX-PC-3) and target-negative cell line (SW-620). Cell death curves are shown separately for each cell line. The mean and standard deviation of the two measurements, as well as the dose-response fitting, are shown. [Figure 31] This study demonstrates dose-dependent induction of signs of DNA damage and apoptosis in response to treatment with escalating concentrations of anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR8. Corresponding non-targeting isotype control conjugates were included as negative controls. MDA-MB-468 cells (5T4 high) were treated with escalating concentrations (0.05–12 μg / ml) of anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR8 or isotype ADC (isotype-P5(PEG24)-VC-PAB-exatecan DAR8) for 72 hours. Cells were stained for cleaved PARP (left), caspase 3 (center), and pH2AX (right) and analyzed by flow cytometry. Graphs show mean (n=2) ± SEM. [Figure 32]This graph shows the binding of the Fc region of anti-TPBG-HC-LALA and anti-TPBG-HC-wt antibodies to recombinant hexameric C1q complement protein, as measured using a human C1q binding assay based on HTRF (homogenous time-resolved fluorescence) (HTRF Human C1q Binding Kit, Cisbio), as described by the manufacturer. In short, serial dilutions from 280 nM to 11.6 nM were measured for all antibodies tested (anti-TPBG mAb HC-wt, anti-TPBG mAb HC-LALA, α-MHC-I positive, IgG1 kit standard). The HTRF ratio was calculated by dividing the acceptor emission signal at 665 nm by the donor emission signal at 620 nm and multiplying by 10000. The graph shows the HTRF ratio at a concentration of 70 nM, after subtracting the background (dilution only) HTRF ratio. The graph shows the mean (n=2) ± SD. [Figure 33]This shows dose-dependent binding of the Fc region of the anti-TPBG-HC-LALA antibody, anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR8 ADC, and anti-TPBG-HC-wt antibody to recombinant human FcRn and FcγR. This was measured using the Lumit® FcγR-binding immunoassay (FcγRn, FcγRI, FcγRIIa / CD32 R131 / H131 polymorphism, FcγRIIIa / CD16 V158 / F158 polymorphism, Promega) according to the manufacturer's instructions. Serial dilutions of anti-TPBG HC-wt, anti-TPBG HC-LALA, and anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR8, standard materials, and trastuzumab as a positive control were incubated with Tracer-LgBiT and FcγR-SmBiT. In the absence of an antibody analyte, or when no interaction occurs between the antibody under test and FcγR, Tracer-LgBiT binds to the FcγR-SmBiT target, resulting in a maximum luminescence signal. When successful interaction with FcγR occurs, the antibody / ADC under test competes with Tracer-LgBiT for binding to the FcγR target, resulting in a concentration-dependent decrease in the luminescence signal. Luminescence was measured using a microplate reader Infinite M200 Pro (Tecan). The graph shows n=1. [Figure 34] This describes an antibody-dependent cytotoxicity (ADCC) assay based on calcein release. Co-cultures of healthy donor (HD) NK cells and calcein-stained target-positive tumor cells (MDA-MB-468 or DU-145) in a 4:1 ratio were incubated with 15 μg / ml of a specified antibody or ADC (anti-MHC-I antibody served as a positive control). Specific cell death rate (%) was calculated by dividing the calcein released by antibody-mediated cell death by the calcein released by cells permeabilized with Triton X (maximum death). The graph shows the mean (n=2) ± SEM. [Figure 35]Figure 35A shows the results of in vivo efficacy analysis of anti-TPBG-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, in a patient-derived head and neck cancer xenograft model (PDX, HN11218). The left side shows the time course of tumor volume after a single treatment on day 0 for various dose levels of anti-TPBG-P5(PEG24)-VC-PAB-exatecan (DAR 8) (1 mg ADC / kg body weight, 3 mg ADC / kg body weight, and 5 mg ADC / kg body weight) and an isotype control with the same linker payload (5 mg / kg), compared to the untreated (vehicle). The right side shows the body weight of animals treated with various dose levels of anti-TPBG-P5(PEG24)-VC-PAB-exatecan (DAR 8) and an isotype control with the same linker payload, compared to the untreated (vehicle). All results are presented as mean values ​​and SEMs for 8 animals per group. Figure 35B shows the in vivo PK evaluation of total and intact ADCs at three dose levels in the dose-response efficacy study, as mean values ​​and SDs of three measurements per time point obtained from mice treated once on day 0 with various dose levels of anti-TPBG-P5(PEG24)-VC-PAB-exatecan (1 mg ADC / kg body weight, 3 mg ADC / kg body weight, and 5 mg ADC / kg body weight). In vivo PK evaluation was performed as described in Example 1. [Figure 36]The results of in vivo efficacy analysis of anti-TPBG-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, in a patient-derived NSCLC xenograft model (PDX, Lu9744) are shown. The left side shows the time course of tumor volume after a single dose of anti-TPBG-P5(PEG24)-VC-PAB-exatecan (DAR 8) (1 mg ADC / kg body weight, 3 mg ADC / kg body weight, and 5 mg ADC / kg body weight) on day 0, compared to the untreated (vehicle) group. The right side shows the body weight of animals treated with anti-TPBG-P5(PEG24)-VC-PAB-exatecan (DAR 8) compared to the untreated (vehicle) group. All results are shown as mean values ​​and SEMs for 4 animals per group. [Modes for carrying out the invention]

[0024] Detailed description of the invention The present invention is described in detail below and is also illustrated by the accompanying embodiments and drawings.

[0025] The inventors have produced and characterized a novel specific anti-TPBG antibody to specifically target the extracellular domain of TPBG. This is particularly advantageous because it relates to a new therapeutic method for treating cancer (for example, preferably the cancer is selected from the group consisting of breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, cervical cancer, rectal cancer, colon cancer, esophageal cancer, gastric cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcoma, brain cancer, nevus and melanoma, genitourinary cancer, prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, cholangiocarcinoma, hematological cancer, retinoblastoma, thyroid cancer, and fallopian tube cancer; more preferably the cancer is a solid tumor, for example, breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, uterine cancer (including, for example, muscular cancer), cervical cancer, rectal cancer). Cancers selected from the group consisting of cancer, colon cancer, anal cancer, esophageal cancer, stomach cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcomas (e.g., osteosarcoma and Kaposi's sarcoma), brain cancer (e.g., pituitary tumors), nevus and melanoma cancer, skin cancer (e.g., squamous cell carcinoma and melanoma), genitourinary cancers (e.g., ureteral and bladder cancer, testicular cancer, prostate cancer, penile cancer), prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancers (e.g., lymphoma, leukemia, myeloma, myelodysplastic syndrome, myelofibrosis), eye cancers (e.g., retinoblastoma), neuroendocrine tumors, and cancers of unknown primary origin (CUP).

[0026] The novel antibody of the present invention is a humanized anti-5T4 monoclonal antibody (mAb) containing Fc silencing mutations such as leucine (L) to alanine (A) substitutions (LALA mutations) at positions 234 and 235.

[0027] The antibody-drug conjugate (ADC) of the present invention, comprising the anti-TPBG antibody of the present invention, may be one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 20, most preferably 4 to 10, most preferably 4 to 10, most preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, Preferably eight cytotoxic moieties (for example, cytotoxic payloads, e.g., tubulin inhibitors, e.g., topoisomerase I inhibitors, e.g., auristatin or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., exatecan) are conjugated preferably via one or more linkers, and more preferably via one or more phosphoamidate linkers.

[0028] definition antibody When used herein, “antibody” may mean a protein comprising one or more polypeptides (including one or more binding domains and / or antigen-binding moieties, preferably antigen-binding domains) substantially or partially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. The term “immunoglobulin” (Ig) is used herein synonymously with “antibody.” Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. In particular, “antibody” as used herein is typically a tetrameric glycosylated protein consisting of two light (L) chains, each about 25 kDa, and two heavy (H) chains, each about 50 kDa. Two types of light chains, called lambda and kappa, may be found in antibodies. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, but IgG is preferred in this invention. Antibodies of this invention having an IgE constant domain or a portion thereof that is bound by the Fc epsilon receptor I are also envisioned. IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called a J chain, containing 10 antigen-binding sites, while IgA antibodies consist of 2 to 5 basic quad-chain units, which can polymerize to form a multivalent aggregate combined with the J chain. In the case of IgG, the quad-chain unit is typically about 150,000 daltons. Each light chain contains a variable (V) domain (VL) and a constant (C) domain (CL) at the N-terminus. Each heavy chain contains an N-terminal V domain (VH), three or four C domains (CH), and a hinge region. While the constant domains are not directly involved in antibody-antigen binding, they can exhibit various effector functions, such as involvement in antibody-dependent cell-mediated cytotoxicity (ADCC).If the antibody needs to exhibit ADCC, it is preferable that it be an IgG1 subtype, as the IgG4 subtype will likely not have the ability to exhibit ADCC.

[0029] The term “antibody” also includes, but is not limited to, monoclonal antibodies, single-specific antibodies; polyspecific or multispecific antibodies such as bispecific antibodies; humanized antibodies, camelized antibodies, human antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, grafted antibodies, and antibodies produced in vitro, with chimeric or humanized antibodies preferred. The term “humanized antibody” is generally defined as an antibody in which a CDR encoding HC and LC specificity is grafted onto a suitable human variable framework ("CDR grafting"). The term “antibody” also includes scFv, single-chain antibodies, diabodies or tetrabodies, domain antibodies (dAb), and nanobodies. From the viewpoint of the present invention, the term “antibody” also includes dimers, trimers, multimers, or bifunctional, trifunctional, or multifunctional antibodies having several antigen-binding sites.

[0030] Furthermore, when used in the present invention, the term “antibody” also refers to derivatives of antibodies (including fragments) as described herein. “Derivatives” of antibodies include amino acid sequences modified by the introduction of substitutions, deletions, or additions of amino acid residues. Furthermore, derivatives also include antibodies modified by covalently bonding any type of molecule to the antibody or protein. Examples of such molecules include, but are not limited to, sugars, PEGs, hydroxyl groups, ethoxy groups, carboxyl groups, or amine groups. In fact, covalent modifications of antibodies result in, but are not limited to, glycosylation, pegylation, acetylation, phosphorylation, and amidation.

[0031] The antibodies of the present invention are preferably “isolated” antibodies. “Isolated,” as used to describe the antibodies disclosed herein, means antibodies identified, separated, and / or recovered from the components of their production environment. Preferably, isolated antibodies are free from all other components originating from their production environment. Contaminating components originating from the production environment, such as those resulting from recombinant transfected cells, are substances that would typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other protein or non-protein solutes. In a preferred embodiment, the antibody is purified to the extent that (1) at least 15 residues of the N-terminal or internal amino acid sequence can be obtained using a spinning cup sequencer, or (2) to the extent that it is considered homogeneous by SDS-PAGE under non-reducing or reducing conditions, using Coomassie blue staining or preferably silver staining. However, typically, isolated antibodies are prepared by at least one purification step.

[0032] The antibodies described herein can be used for diagnostic purposes, including sample testing and in vivo imaging, for which antibodies (or their conjugated fragments) can be conjugated to appropriate detectable substances to form immunoconjugates. For diagnostic purposes, appropriate substances are detectable labels, including radioisotopes for whole-body imaging, and radioisotopes, enzymes, fluorescent labels, and other appropriate antibody tags for sample testing. Detectable labels can be any of the various types currently used in the field of in vitro diagnostics, including particulate labels containing metal sols such as colloidal gold; isotopes; chromophores including fluorescent markers, biotin, luminescent markers, and phosphorescent markers; and enzymatic labels that convert a given substrate into a detectable marker; as well as polynucleotide tags that become apparent after amplification by polymerase chain reaction, etc. In this case, biotinylated antibodies would be detectable by avidin conjugation or streptavidin conjugation. Appropriate enzymatic labels include horseradish peroxidase and alkaline phosphatase, etc. For example, the label may be an alkaline phosphatase enzyme, or a suitable lanthanide chelate such as terbium(III) and europium(III), which is detected by measuring the presence or formation of chemiluminescence after conversion of a 1,2-dioxetane substrate (e.g., adamantylmethoxyphosphoryloxyphenyldioxetane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}decane}-4-yl)phenyl phosphate disodium (CSPD), and CDP and CDP-star®), or other luminescent substrates well known to those skilled in the art, such as terbium(III) and europium(III). The detection means will depend on the selected label. The appearance of the label or its reaction product can be observed visually if the label is particulate and accumulates at an appropriate level, or it can be observed using instruments such as a spectrophotometer, illuminometer, and fluorometer, in either case according to standard practices.

[0033] The "effector function" of an antibody refers to a biological activity attributable to the Fc region of the antibody (native sequence Fc region or amino acid sequence variant Fc region), which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation. Antibodies recruit effector cells, so to speak, to exert their effector function.

[0034] As used herein, the term "antigen-binding portion" means a fragment of an immunoglobulin (or intact antibody) and encompasses any polypeptide that includes an antigen-binding fragment or antigen-binding domain. Preferably, the fragment is, for example, Fab, F(ab'), F(ab')2, Fv, scFv, Fd, disulfide-bonded Fv (sdFv), and other antibody fragments that retain the antigen-binding function described herein. Typically, such fragments contain an antigen-binding domain and are expected to have the same properties as the antibodies described herein. Thus, the fragment can preferably also bind to the extracellular domain of TPBG.

[0035] As used herein, the term "specifically binds" means an antibody or fragment or derivative thereof that specifically binds to the TPBG protein and does not specifically bind to another protein. The antibodies or fragments or derivatives thereof according to the invention bind to the TPBG protein via the variable domain of the antibody.

[0036] The VH and VL domains, paired together, jointly form a single antigen-binding site. The CH domain closest to the VH is called CH1. Each L chain is linked to the H chain by a single disulfide covalent bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The VH and VL domains consist of four regions with relatively conserved sequences called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions with hypervariable sequences (complementarity-determining regions, CDRs). The CDRs contain most of the residues responsible for the specific interaction between the antibody and the antigen. The CDRs are called CDR1, CDR2, and CDR3. Therefore, the CDR components on the heavy chain are called H1 or H-CDR1 (or CRD-H1), H2 or H-CDR2 (or CDR-H2), and H3 or H-CDR3 (or CDR-H3), while the CDR components on the light chain are called L1 or L-CDR1 (or CRD-L1), L2 or L-CDR2 (or CDR-L2), and L3 or L-CDR3 (or CDR-L3).

[0037] The term "variable" refers to a portion of the immunoglobulin domain (i.e., the "variable domain") that exhibits sequence variability and is involved in determining the specificity and binding affinity of individual antibodies. Variability is not evenly distributed throughout the antibody's variable domain, but rather concentrated in the subdomains of the heavy chain and light chain variable regions. These subdomains are called "complementarity-determining regions" (CDRs).

[0038] The terms “CDR” and its plural form “CDRs” refer to complementarity-determining regions (CDRs), three of which form binding features for the light chain variable region (L1-CDR, L2-CDR, and L3-CDR), and three which form binding features for the heavy chain variable region (H1-CDR, H2-CDR, and H3-CDR). CDRs contribute to the functional activity of antibody molecules and are separated by amino acid sequences that constitute scaffolding or framework regions. The precise definitional boundaries and lengths of CDRs vary depending on the classification and numbering scheme. Therefore, CDRs may be referred to based on any other boundary definition, including Kabat, Chothia, contact definition, or any other numbering scheme described herein. Although the boundaries differ, each of these schemes has some overlap in the elements that constitute the so-called “hypervariable regions” within the variable sequence. Therefore, definitions of CDRs based on these schemes may differ in terms of length and the boundary regions with adjacent framework regions. However, numbering according to the so-called Kabat scheme is preferred.

[0039] The highly conserved (i.e., non-hypervariable) portions of the variable domain are called "framework" regions (FRMs). The variable domains of the natural heavy and light chains each contain four FRM regions, which primarily adopt a β-sheet configuration and are linked by three hypervariable regions. These hypervariable regions form loops, linking the β-sheet structure and, in some cases, forming part of it. The hypervariable regions of each chain are brought together very closely by the FRMs and, together with the hypervariable region of the other chain, contribute to the formation of the antigen-binding surface (see Kabat et al., above). The constant domains do not directly participate in antigen binding but exhibit various effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement activation.

[0040] The term “binding domain” in relation to the present invention characterizes a domain of a polypeptide that specifically binds to / interacts with a given target epitope. “Epitope” is antigenic, and therefore the term epitope may also be referred to herein as “antigenic structure” or “antigenic determinant.” Thus, the binding domain is an “antigen interaction site.” According to the present invention, the term “antigen interaction site” defines a polypeptide motif that can specifically interact with a particular antigen or a particular group of antigens, such as the same antigen in different species. It is also understood that this binding / interaction constitutes “specific recognition.”

[0041] The terms “antigen-binding domain,” “antigen-binding portion,” “antigen-binding fragment,” and “antibody-binding region,” as used herein, refer to a portion of an antibody molecule containing amino acids responsible for the specific binding of an antibody to an antigen. The portion of the antigen that is specifically recognized and bound by the antibody is referred to herein as an “epitope,” as previously stated. As previously stated, the antigen-binding domain may typically include the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH).

[0042] The term “epitope” also means a site on an antigen (in the context of this invention, the antigen is a TPBG protein) to which an antibody molecule binds. Preferably, an epitope is a site on a molecule (in the context of this invention, the antigen is a TPBG protein) to which an antibody or its antigen-binding moiety, preferably an antibody, is produced and / or which the antibody binds. For example, an epitope can be recognized by an antibody or its antigen-binding moiety. A “linear epitope” is an epitope whose recognized epitope is composed of a primary amino acid sequence. Typically, a linear epitope contains at least three, and more commonly at least five, for example, about eight to about ten amino acids, in a unique sequence.

[0043] The term "cross-reactivity" can refer to the ability of an antibody to react with similar antigenic sites on different proteins.

[0044] In this context, the term "specific" may mean that an antibody or its antigen-binding moiety binds to a target TPBG but not to another protein. The term "another protein" includes any protein in which the antibody or its antigen-binding moiety is closely related to or homologous to the target TPBG protein. However, the term "another protein" does not include cross-reactivity of an antibody or its antigen-binding moiety with a TPBG protein originating from a different species than the one in which the antibody or its antigen-binding moiety was produced.

[0045] Therefore, a species-cross-reactive specific antibody or its antigen-binding moiety targeting the TPBG protein is preferably intended by the present invention.

[0046] "K D The term "k" refers to the equilibrium dissociation constant, i.e., k, between an antibody and its antigen, or between the variable regions of one heavy chain and one light chain of an antibody or its fragment or derivative and their antigens. off / k on This can mean a ratio; where the antigen is, for example, TPBG, for example, full-length TPBG and / or one or more fragments thereof, preferably the one or more fragments comprising at least one extracellular domain (ECD) of the TPBG (for example, the ECD contains at least 32-355 amino acids of human TPBG having Uniprot accession number: Q13641 or SEQ ID NO: 1) and / or one or more fragments of the ECD (for example, having a length of about 15-30 amino acids); K D This is measured in vitro. D The affinity is inversely proportional to the affinity.

[0047] As used herein, the term "affinity" may mean the binding strength between the variable regions of one heavy chain and one light chain of an antibody or a fragment or derivative thereof and their antigen; where the antigen is, for example, TPBG, for example, full-length TPBG and / or one or more fragments thereof, preferably the one or more fragments comprising at least one extracellular domain (ECD) of the TPBG (e.g., the ECD contains at least 32-355 amino acids of human TPBG having Uniprot accession number: Q13641 or SEQ ID NO: 1) and / or one or more fragments of the ECD (e.g., having a length of about 15-30 amino acids); affinity is measured in vitro. Affinity determines the strength of the interaction between the epitope and the antigen-binding site of the antibody. Affinity can be calculated using the following formula. KA = [AB - AG] / [AB] × [AG] = k on / k off During the ceremony, KA = affinity constant [AB] = Molar concentration of vacant binding sites on the antibody [AG] = Molar concentration of vacant binding sites on the antigen [AB-AG] = Molar concentration of antibody-antigen complex

[0048] The terms “amino acid” or “amino acid residue” typically mean an amino acid selected from the group consisting of amino acids having the definitions accepted in the art, such as alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified amino acids, synthetic amino acids, or rare amino acids may also be used as desired. Generally, amino acids can be grouped into those having nonpolar side chains (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val); charged side chains (e.g., Asp, Glu); positively charged side chains (e.g., Arg, His, Lys); or uncharged side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0049] The term “polypeptide” is used herein as equivalent to the term “protein.” Proteins (including their fragments, preferably biologically active fragments, and peptides, which typically have fewer than 30 amino acids) contain one or more amino acids linked to one another via covalent peptide bonds (which give rise to chains of amino acids). When used herein, the term “polypeptide” refers, for example, to a group of molecules consisting of more than 30 amino acids. Polypeptides may also form multimers, i.e., multiple polypeptide molecules, such as dimers, trimers, and higher-order oligomers. The polypeptide molecules forming such dimers, trimers, etc., may be identical or non-identical. As a result, the corresponding higher-order structures of such multimers are called homodimers or heterodimers, homotrimers or heterotrimers, etc. An example of a heteromultimer is the antibody molecule, which in its natural form consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms “polypeptide” and “protein” also refer to naturally modified polypeptides / proteins that are modified by post-translational modifications such as glycosylation, acetylation, and phosphorylation, for example. Such modifications are well known in the art.

[0050] The term "immune cells" refers to cells capable of producing antibodies. Immune cells of particular interest in this specification are, for example, lymphoid cells derived from the spleen, peripheral blood lymphocytes (PBLs), lymph nodes, inguinal lymph nodes, Peyer's patches, tonsils, bone marrow, umbilical cord blood, pleural fluid, and tumor-infiltrating lymphocytes (TILs).

[0051] One type of antibody variant encompassed by the present invention is an amino acid substitution variant. In these variants, at least one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues in the TPBG antibody molecule are substituted with other residues. The sites of greatest interest for substitutional mutagenesis include the CDRs of the heavy chain and / or light chain, specifically the hypervariable region, but alterations of the FRs of the heavy chain and / or light chain are also intended.

[0052] For example, if a CDR sequence contains 6 amino acids, it is expected that one, two, or three of these amino acids may be substituted. Similarly, if a CDR sequence contains 15 amino acids, it is expected that one, two, three, four, five, or six of these amino acids may be substituted.

[0053] Generally, when amino acids are substituted in one, more, or all of the heavy chain and / or light chain CDRs, it is preferable that the resulting "substituted" sequence is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), more preferably 65%, even more preferably 70%, particularly preferably 75%, and most preferably 80% identical to the "original" CDR sequence. This means that the degree to which a CDR is identical to the "substituted" sequence depends on the length of the CDR. For example, a CDR with 5 amino acids is preferably 80% identical to its substituted sequence in order that at least one amino acid is substituted. Therefore, the CDRs of a TPBG antibody may have varying degrees of identity with respect to their substituted sequences; for example, CDRL1 may have 80% identity, while CDRL3 may have 90% identity.

[0054] A preferred substitution (or replacement) is a conservative substitution. However, any substitution (including non-conservative substitutions or one or more of the “exemplary substitutions” listed in Table I herein) is considered, as long as the antibody retains its ability to specifically bind to the TPBG protein and / or its CDR has identity to the substituted sequence (at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), more preferably 65%, even more preferably 70%, particularly preferably 75%, and even more preferably 80% identity to the “original” CDR sequence).

[0055] Conservative substitutions are listed under the “Preferred Substitutions” section in Table I. If such substitutions alter biological activity, more substantial changes may be introduced, referred to as “Exemplary Substitutions” in Table I or further described later with respect to amino acid classes, and the products may be screened for desired characteristics.

[0056] chemical modification The antibody or its antigen-binding variant or fragment used in accordance with the present invention may be modified. Typical modifications that may be considered in the context of the present invention include, for example, the chemical modifications described below.

[0057] Possible chemical modifications to antibodies or their antigen-binding variants or fragments include acylation or acetylation of the amino terminus, or amidation or esterification of the carboxyl terminus, or such modifications at both ends. Modifications may also affect the amino group of the lysine side chain or the hydroxyl group of threonine. Other suitable modifications include, for example, extension of the amino group by polypeptide chains of varying lengths (e.g., XTEN technology or PASylation®), N-glycosylation, O-glycosylation, and chemical bonding of carbohydrates, e.g., hydroxyethyl starch (e.g., HESylation®) or polysialic acid (e.g., PolyXen® technology). Chemical modifications such as alkylation (e.g., methylation, propylation, butylation), arylation, and etherification may also be possible and are also envisioned.

[0058] Antibody-drug conjugates (ADCs) The term antibody-drug conjugate (or ADC), as used herein, may mean any antibody according to the present invention conjugated to one or more drug moieties (e.g., cytotoxic payloads). Preferably, the antibody-drug conjugate (ADC) of the present invention comprises one or more anti-TPBG antibodies of the present invention (e.g., humanized monoclonal TPBG-specific IgG1 antibodies) conjugated to a cytotoxic payload, wherein (a) the cytotoxic payload is selected from the group consisting of camptothecin, mytansinoids, calicheamycin, duochamycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof; and / or (b) the cytotoxic payload is exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, berotecan, lulu (c) The cytotoxic payload is a camptothecin moiety C selected from the group consisting of totecan, rubitecan, silatecan, cositecan, and gimatecan; and / or (c) the cytotoxic payload is conjugated via a cleavable linker (L), preferably the linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction, and more preferably the linker is cleavable by a protease, preferably a cathepsin such as cathepsin B; and / or (d) the linker (L) comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) the cytotoxic payload is an exatecan conjugated via a chemical valine-citrulline-PAB releasing unit or a valine-alanine-PAB releasing unit, the releasing unit being cleavable by a protease.As used herein, “linker” (L) may mean any chemical moiety capable of linking the antibody of the present invention to one or more drug moieties (e.g., cytotoxic moieties (e.g., cytotoxic payloads, e.g., tubulin inhibitors, e.g., topoisomerase-I inhibitors, e.g., auristatin or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., exatecan)). Preferably, L is a phosphoamidate linker; more preferably, linker L comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; most preferably, linker L is cleavable (e.g., readily cleaved by enzymes).

[0059] Sequence identity The terms "identity percentage (%)" or "sequence identity percentage (%)", as used in this invention, may mean the percentage of identical pairs of residues relative to the number of residues in the longer of the two sequences, after the polypeptide sequence of the invention has been (homologically) aligned with the sequence in question. The identity percentage is obtained by dividing the number of identical residues by the total number of residues and multiplying the result by 100.

[0060] The percentage of sequence homology or sequence identity can be determined herein, for example, using BLASTP, version blastp 2.2.5 (November 16, 2002; Altschul, SF et al. (1997) Nucl. Acids Res. 25, 3389-3402). In this embodiment, the percentage of homology is preferably based on the alignment of the entire polypeptide sequence including the propeptide sequence (matrix: BLOSUM 62; gap cost: 11.1) using the wild-type protein backbone as a reference in pairwise comparison. This is calculated as the percentage obtained by dividing the number of "positive" (homologous amino acids) shown as a result in the BLASTP program output by the total number of amino acids selected by the program for alignment.

[0061] The term "TPBG" refers to carcinoembryonic antigen 5T4 (also known as trophoblast glycoprotein (TPBG), and both terms may be used interchangeably herein), and typically includes all known isoforms. Preferably, this TPBG is human TPBG having SEQ ID NO:1 or UniProtKB accession number Q13641 or SEQ ID NO:1.

[0062] vector The nucleic acids of the present invention may also be in the form of a vector, present within a vector, and / or be part of a vector.

[0063] The term "vector" refers to a nucleic acid molecule used as a carrier for transferring (foreign) genetic material into a host cell, and non-limitingly includes plasmids, viruses, cosmids, and artificial chromosomes, such as bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Typically, a designed vector includes an origin of replication, a multicloning site, and a selection marker. Typically, the vector itself is a nucleotide sequence, usually a DNA sequence, containing an insertion fragment (transgene) and a larger sequence that serves as the vector's "backbone." In addition to the transgene insertion fragment and backbone, a vector may include additional elements, such as gene regulatory elements, gene markers, antibiotic resistance, reporter genes, targeting sequences, or protein purification tags. Specifically envisioned in the context of this invention are expression vectors (expression constructs) for the expression of a transgene in a host cell, which typically include a gene regulatory sequence in addition to the transgene.

[0064] Generally, an expression vector is a vector capable of expressing the antibody of the present invention in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Those skilled in the art will readily understand that the selection of a particular vector is determined by, for example, the host cell, the intended copy number of the vector, and whether transient or stable expression of the antibody of the present invention is envisioned.

[0065] "Transient expression" occurs when nucleic acids (e.g., linear or nonlinear DNA or RNA molecules) or vectors that are incapable of autonomous replication are introduced into recipient host cells. The expression of the introduced gene occurs through the transient expression of the introduced sequence.

[0066] However, the “stable expression” of the nucleic acid sequences described herein is often preferred, which can be achieved by stably incorporating the nucleic acid sequences into the genome of a host cell, or by introducing a vector containing the nucleic acid sequences of the present invention that can autonomously replicate into a host cell.

[0067] The vectors provided herein are particularly intended to include gene regulatory elements functionally linked to the DNA sequence encoding the antibody of the present invention.

[0068] The term “gene regulatory element” refers to DNA sequences required for the expression of functionally linked coding sequences within a particular host organism. The term “gene regulatory element” includes other elements that can control gene expression, including controllable transcription promoters, operators, enhancers, silencers, transcription terminators, 5' and 3' untranslated regions, and start and end codons, which interact with host cellular proteins to perform transcription and translation. The exact nature of the regulatory regions required for gene expression can vary from organism to organism. Prokaryotic gene regulatory elements include, for example, promoters, optionally operator sequences, and ribosome-binding sites (RBS), while eukaryotic gene regulatory elements include promoters, polyadenylation (poly-A) signals, and enhancers.

[0069] Gene regulatory elements are assumed to be "functionally linked" to the gene they are intended to express, that is, positioned to functionally relate to that gene. For example, a promoter or enhancer is "functionally linked" to a coding nucleic acid sequence if it affects the transcription of that sequence. Functionally linked DNA sequences may or may not be adjacent. Typically, linking is achieved by ligation at a convenient restriction site or by synthetic oligonucleotide adapters or linkers.

[0070] host cell Furthermore, host cells containing the vectors described herein (e.g., recombinant host cells and / or isolated host cells) are also provided herein.

[0071] Various host cells can be used to express nucleic acid sequences encoding the antibodies described herein. Host cells can be prepared using genetic engineering methods known in the art. The process of introducing a vector into recipient host cells is also referred to below as "transformation" or "transfection." These terms are used synonymously herein.

[0072] Host cell transformation typically involves creating transient pores or "holes" in the cell wall and / or cell membrane to allow uptake of substances. Exemplary transformation protocols include the use of calcium phosphate, electroporation, cell compression, dendrimers, liposomes, cationic polymers such as DEAE-dextran or polyethyleneimine, sonoporation, optical transfection, impalefection, nanoparticles (gene guns), magnetofection, microparticle guns, alkaline cations (cesium, lithium), enzymatic digestion, agitation with glass beads, or viral vectors. The choice of method usually depends on the type of cell being transformed, the vector introduced into the cell, and the conditions under which the transformation takes place.

[0073] As used herein, the term “host cell” may mean any cell or cell culture that serves as the recipient of an antibody (Ab) encoding vector or isolated nucleic acid sequence as described herein. Suitable host cells include, but are not limited to, prokaryotic or eukaryotic cells, as well as bacteria, yeast cells, fungal cells, plant cells, and animal cells, such as insect cells and mammalian cells, such as mouse, rat, macaque, or human cells.

[0074] For example, antibodies (Ab) can be produced within bacteria. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable hosts for cloning or expression of the TPBG antibodies of the present invention. Exemplary examples include hosts of the genus Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces, such as K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 36906), K. thermotolerance, and K. marxianus; and the genus Yarrowia (EP 402). This includes 226); Pichia pastoris (EP 183 070); Candida; Trichoderma reesia (EP 244 234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and hosts of filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus, e.g., A. nidulans and A. niger.

[0075] Host cells suitable for the expression of the glycosylated antibody constructs of the present invention may also be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants, as well as corresponding permissible insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm), have been identified. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of silkworm NPV, are publicly available.

[0076] Plant cell cultures of cotton, maize, potato, soybean, petunia, tomato, Arabidopsis, and tobacco can also be used as hosts. Cloning vectors and expression vectors useful for protein production in plant cell cultures are known to those skilled in the art.

[0077] Examples of useful mammalian host cell lines include: SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human fetal kidney cell line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO), mouse Sertoli cells (TM4); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, 1413 8065); mouse mammary tumor cells (MMT 060562, ATCC These include CCL51 cells, TRI cells, MRC 5 cells, FS4 cells, and human hepatocellular carcinoma cells (Hep G2).

[0078] patient Where used herein, the terms “patient” or “subject” mean human or non-human animals, usually mammals. Specifically, mammals are assumed to be, for example, rabbits, mice, rats, guinea pigs, hamsters, dogs, cats, pigs, cows, goats, sheep, horses, monkeys, apes, or preferably humans. Thus, the methods, uses, and compounds described in this document are generally applicable to both human and veterinary diseases.

[0079] treatment The term “treatment” in all its grammatical forms includes therapeutic or preventive treatments. “Therapeutic or preventive treatments” include preventive treatments aimed at complete prevention of clinical and / or pathological symptoms, or therapeutic treatments aimed at improvement or remission of clinical and / or pathological symptoms of a disease. Therefore, the term “treatment” also includes improvement or prevention of cancer.

[0080] In the present invention, the term “therapeutic effect” usually means a desirable or beneficial effect of treatment, e.g., improvement or remission of disease symptoms. The term “symptoms” of a disease is used herein to describe its perceptible manifestation, and this term includes both clinical symptoms (defined below as signs of the disease that may be detected during a physical examination and / or perceptible to the patient (i.e., subjective symptoms)) and pathological symptoms, meaning the manifestation of the disease at the cellular and molecular levels. The therapeutic effect of treatment with the TPBG-ADC of the present invention can be evaluated using routine methods in the art, for example, by measuring leukemia burden by blood / bone marrow analysis (cytomorphology, flow cytometry, genetics), clinical chemistry, or radiological techniques (e.g., CT). Alternatively, the overall condition of each patient (e.g., health status, well-being) can also be evaluated, which also helps a skilled physician to assess whether a therapeutic effect has been elicited. Those skilled in the art know of numerous other methods suitable for observing the therapeutic effect of the compounds of the present invention.

[0081] dose Preferably, a therapeutically effective dose of the compound described herein is administered. “Therapeutically effective dose” means the amount of the compound described herein that elicits a therapeutic effect. The precise dose of the Ab-TPBG-ADC of the present invention depends on the purpose of the treatment (e.g., induction or maintenance of remission) and can be determined by those skilled in the art using known techniques. Adjustments may be necessary depending on the route of administration, age, weight, overall health, sex, diet, administration time, drug interactions, and severity of the condition, which can be determined by those skilled in the art using routine experimental methods.

[0082] Administration Various routes, including but not limited to oral, topical, transdermal, subcutaneous, intravenous, intraperitoneal, intramuscular, or intraocular, preferably subcutaneous and / or intravenous, are applicable to the administration of the compounds according to the present invention. However, if desired, those skilled in the art can readily select any other route.

[0083] composition The TPBG antibody and / or ADC of the present invention are intended to be administered in the form of a pharmaceutical composition.

[0084] The term "pharmaceutical composition" specifically means a composition suitable for administration to humans, i.e., a composition that is preferably sterile and / or contains pharmaceutically acceptable components. However, compositions suitable for administration to non-human animals are also assumed herein. Preferably, a pharmaceutical composition comprises the Ab-TPBG-ADC of the present invention together with one or more pharmaceutically acceptable excipients. The term "excipients" includes bulking agents, binders, disintegrants, coating agents, adsorbents, anti-adhesion agents, flow enhancers, preservatives, antioxidants, flavoring agents, colorants, sweeteners, solvents, auxiliary solvents, buffers, chelating agents, viscosity modifiers, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers, or osmotic pressure modifiers. The pharmaceutical compositions of the present invention can be formulated in various forms, for example, as solids, liquids, gases, or lyophilized forms; in particular, they may be in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures, or fluid extracts, or in forms particularly suitable for a desired method of administration.

[0085] The pharmaceutical composition of the present invention may further contain one or more additional active ingredients. Preferably, these active ingredients are therapeutically effective for treating the diseases described herein and are present in the composition in therapeutically effective amounts.

[0086] Accordingly, in view of the foregoing, the present invention also provides a pharmaceutical composition comprising one or more TPBG antibodies and / or ADCs of the present invention. This pharmaceutical composition is particularly intended for use in methods of therapeutic and / or prophylactic treatment of cancer.

[0087] kit Kits are also provided herein. The kit may be a kit consisting of two or more parts, and the kit preferably contains the TPBG antibody and / or ADC of the present invention in a therapeutically effective amount and in a pharmaceutically acceptable form. The components of the kit may be placed in a container or vial. The kit is envisioned to contain additional agents useful for the treatment of cancer, as described elsewhere herein.

[0088] Chemical group Unless otherwise defined, the term "alkyl", by itself or as part of another term, generally means a substituted or unsubstituted straight-chain or branched-chain saturated hydrocarbon having the specified number of carbon atoms; for example, "-(C1-C8)alkyl" or "-(C1-C 10 )alkyl" each mean an alkyl group having 1 to 8 or 1 to 10 carbon atoms, respectively. When the number of carbon atoms is not specified, the alkyl group may have 1 to 8 carbon atoms. Representative straight-chain -(C1-C8)alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, and -n-octyl, and branched-chain -(C1-C8)alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl. In some aspects, the alkyl group may be unsubstituted. Optionally, the alkyl group may be substituted with, for example, one or more groups.

[0089] Unless otherwise defined, the term "alkylene", by itself or as part of another term, generally means the stated number of carbon atoms, preferably 1 to 10 carbon atoms (-(C1-C 10This refers to a substituted or unsubstituted branched or linear saturated hydrocarbon radical having 1 to 8 carbon atoms (-(C1-C8)alkylene-) or preferably 1 to 8 carbon atoms (-(C1-C8)alkylene-), and having two monovalent radical centers resulting from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkane. If the number of carbon atoms is not specified, the alkylene group may have 1 to 8 carbon atoms. Typical alkylene radicals include, but are not limited to, methylene (-CH2-), 1,2-ethylene (-CH2CH2-), 1,3-n-propylene (-CH2CH2CH2-), and 1,4-n-butylene (-CH2CH2CH2CH2-). In some aspects, the alkylene group may not be substituted. Optionally, the alkylene group may be substituted, for example, with one or more groups.

[0090] Unless otherwise specified, the term “alkenyl” generally means, either by itself or as part of another term, a substituted or unsubstituted straight-chain or branched-chain unsaturated hydrocarbon having a double bond and a specified number of carbon atoms; for example, “-(C2~C8)alkenyl” or “-(C2~C 10 The term "(C2-C8) alkenyl" refers to an alkenyl group having 2 to 8 or 2 to 10 carbon atoms, respectively. If the number of carbon atoms is not specified, the alkenyl group may have 2 to 8 carbon atoms. Representative (C2-C8) alkenyl groups include, but are not limited to, -ethenyl, -1-propenyl, -2-propenyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, and -2,3-dimethyl-2-butenyl. In some cases, the alkenyl group may not be substituted. Optionally, the alkenyl group may be substituted with, for example, one or more groups.

[0091] Unless otherwise specified, the term "alkenylene" means, by itself or as part of another term, generally a number of carbon atoms, preferably 2 to 10 carbon atoms (-(C2-C 10 The term "alkenylene" refers to a substituted or unsubstituted branched or straight-chain unsaturated hydrocarbon radical having 2 to 8 carbon atoms (-(C2-C8)alkenylene-) or preferably 2 to 8 carbon atoms, having a double bond, and having two monovalent radical centers resulting from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkene. If the number of carbon atoms is not specified, the alkenylene group may have 2 to 8 carbon atoms. Typical alkenylene radicals include, but are not limited to, -ethenylene-, -1-propenylene-, 2-propenylene-, -1-butenylene-, -2-butenylene-, -isobutenylene-, -1-pentenylene-, -2-pentenylene-, -3-methyl-1-butenylene-, -2-methyl-2-butenylene-, and -2,3-dimethyl-2-butenylene-. In some cases, the alkenylene group may not be substituted. Optionally, the alkenylene group may be substituted, for example, with one or more other groups.

[0092] Unless otherwise specified, the term “alkynyl” generally means, either by itself or as part of another term, a substituted or unsubstituted straight-chain or branched-chain unsaturated hydrocarbon having a triple bond and a specified number of carbon atoms; for example, “-(C2~C8)alkynyl” or “-(C2~C 10The term "(C2-C8) alkynyl" refers to an alkynyl group having 2 to 8 or 2 to 10 carbon atoms, respectively. If the number of carbon atoms is not specified, the alkynyl group may have 2 to 8 carbon atoms. Representative -(C2-C8) alkynyl groups include, but are not limited to, -acetylenyl, -1-propynyl, -2-propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1-butynyl. In some cases, the alkynyl group may not be substituted. Optionally, the alkynyl group may be substituted with, for example, one or more groups.

[0093] Unless otherwise specified, the term "alkynylene" means, by itself or as part of another term, generally a number of carbon atoms as described, preferably 2 to 10 carbon atoms (-(C2-C 10 This refers to a substituted or unsubstituted branched or straight-chain unsaturated hydrocarbon radical having 2 to 8 carbon atoms (-(C2-C8)alkylylene-) or preferably 2 to 8 carbon atoms, having a triple bond, and having two monovalent radical centers resulting from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. If the number of carbon atoms is not specified, the alkylylene group may have 2 to 8 carbon atoms. Typical alkylylene radicals include, but are not limited to, -ethynylene-, -1-propynylene-, -2-propynylene-, -1-butynylene-, -2-butynylene-, -1-pentynylene-, -2-pentynylene-, and -3-methyl-1-butynylene-. In some aspects, the alkylylene group may not be substituted. Optionally, the alkylylene group may be substituted, for example, with one or more groups.

[0094] Unless otherwise specified, the term “aryl” generally refers, either by itself or as part of another term, to a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical having 6 to 20 carbon atoms (preferably 6 to 14 carbon atoms, more preferably 6 to 10 carbon atoms, and in a very preferred embodiment, 6 carbon atoms) resulting from the removal of one hydrogen atom from one carbon atom of an aromatic ring system. Some aryl groups are represented as “Ar” in exemplary structures. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzenes, naphthalenes, anthracenes, and biphenyls. An exemplary aryl group is the phenyl group. In some aspects, aryl groups may not be substituted. Optionally, aryl groups may be substituted, for example, with one or more groups.

[0095] Unless otherwise specified, the term "arylene" generally refers, either by itself or as part of another term, to the aryl group as defined above, in which one of the hydrogen atoms of the aryl group is replaced by a bond (i.e., divalent), with the following structure when phenyl is an exemplary group: As shown in TIFF2026513098000001.tif22128, it can take the configurations of para, meta, or ortho.

[0096] In a selected embodiment, the arylene is the aryl group defined above, in which two or more of the hydrogen atoms of the aryl group are replaced by bonds (i.e., the arylene may be trivalent). In some aspects, the arylene group may not be substituted. Optionally, the alkynylene group may be substituted, for example, with one or more groups.

[0097] Unless otherwise specified, the terms “heterocycle” or “heterocycle” generally refer to a specified number of carbon atoms, either by themselves or as part of another term (e.g., “(C3-C8) heterocycle” or “(C3-C8) heterocycle”). 10A heterocycle means a heterocycle having 3 to 8 or 3 to 10 carbon atoms, and a monocyclic or bicyclic system having 1 to 4 heteroatom ring members independently selected from N, O, P, or S, and resulting from the removal of one hydrogen atom from the ring atoms of the parent ring system, and being monovalent, substituted or unsubstituted aromatic or non-aromatic. One or more N, C, or S atoms in a heterocycle can be oxidized. A ring containing heteroatoms can be aromatic or non-aromatic. Unless otherwise noted, a heterocycle is bonded to its pendant group at any heteroatom or carbon atom that results in a stable structure. Representative examples of (C3-C8) heterocycles include, but are not limited to, pyrrolidinyl, azetidinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiophene), furanyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridadinyl, isothiazolyl, and isoxazolyl. In some aspects, the heterocyclic group does not need to be substituted. Optionally, the heterocyclic group may be substituted with, for example, one or more groups.

[0098] Unless otherwise specified, the terms “heterocyclo” or “heterocyclic” generally refer, either by themselves or as part of another term, to a heterocyclic group having the number of carbon atoms defined above and specified (e.g., (C3-C8) heterocyclic or (C3-C8) heterocyclic). 10 A heterocyclic group is a heterocyclic group in which one of the hydrogen atoms of the heterocyclic group is replaced by a bond (i.e., it is divalent). In a selected embodiment, a heterocyclo is a heterocyclic group as defined above in which two or more of the hydrogen atoms of the heterocyclic group are replaced by bonds (i.e., the heterocyclo may be trivalent). In some aspects, a heterocyclo or heterocyclic group may not be substituted. Optionally, a heterocyclo or heterocyclic group may be substituted, for example, with one or more groups.

[0099] Unless otherwise specified, the terms “carbocyclic” or “cyclic carbon” generally refer to a ring having a specified number of carbon atoms, either by itself or as part of another term, resulting from the removal of one hydrogen atom from the ring atoms of a parent ring system (e.g., “(C3-C8) carbocyclic” or “(C3-C8) 10 A "carbocyclic ring" means a monovalent, substituted or unsubstituted aromatic or unaromatic, monocyclic or dicyclic cyclic carbon system (meaning a carbocyclic ring having 3 to 8 or 3 to 10 carbon atoms). Exemplary but non-limiting examples include 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, or 8-membered carbocyclic rings. Representative (C3-C8) carbocyclic rings include, but are not limited to, phenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrielinyl, cyclooctyl, and cyclooctadienyl. In some aspects, carbocyclic rings do not need to be substituted. Optionally, carbocyclic rings may be substituted, for example, with one or more groups.

[0100] Unless otherwise specified, the terms “carbocyclo” or “cyclic carbon” generally refer, either by themselves or as part of another term, to a carbon atom having the number of carbon atoms defined above (for example, “(C3-C8) carbocyclo” or “(C3-C8) 10"Carbocyclo" means a carbocyclo or cyclic carbon having 3 to 8 or 3 to 10 carbon atoms respectively, wherein one of the hydrogen atoms of the carbocyclo is replaced by a bond (i.e., it is divalent). In a selected embodiment, the carbocyclo or cyclic carbon is the carbocyclo defined above, wherein two or more of the hydrogen atoms of the carbocyclo are replaced by bonds (i.e., the carbocyclo or cyclic carbon may be trivalent). In some aspects, the carbocyclo or cyclic carbon may not be substituted. Optionally, the heterocyclo or heterocycle may be substituted, for example, with one or more groups.

[0101] Unless otherwise specified, the term "heteroalkyl" by itself or in combination with other terms means a stable straight-chain or branched-chain hydrocarbon or combination thereof, which is fully saturated or contains 1 to 3 degrees of unsaturation and the number of carbon atoms specified (e.g., (C1-C8) heteroalkyl or (C1-C8) 10The heteroalkyl group consists of a heteroalkyl group and 1 to 10, preferably 1 to 3, heteroatoms selected from the group consisting of O, N, Si, and S, where the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, and S may be located at any internal position of the heteroalkyl group or at a position where the alkyl group is bonded to the rest of the molecule. The heteroatom Si may be located at any position of the heteroalkyl group, including a position where the alkyl group is bonded to the rest of the molecule. Examples include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -NH-CH2-CH2-NH-C(O)-CH2-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=NO-CH3, and -CH=CH-N(CH3)-CH3. For example, up to two heteroatoms may be consecutive, such as -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. In preferred embodiments, the (C1-C4) heteroalkyl or heteroalkylene has 1-4 carbon atoms and 1 or 2 heteroatoms, and the (C1-C3) heteroalkyl or heteroalkylene has 1-3 carbon atoms and 1 or 2 heteroatoms. In some aspects, the heteroalkyl or heteroalkylene is saturated. In some aspects, the heteroalkyl or heteroalkylene may be unsubstituted. Optionally, the heteroalkyl or heteroalkylene may be substituted, for example, with one or more groups.

[0102] Unless otherwise specified, the term “heteroalkylene” means a divalent group derived from a heteroalkyl (as described above) having a specified number of carbon atoms, either by itself or as part of another substituent (e.g., (C1-C8)heteroalkylene or (C1-C 10Examples of heteroalkylenes are -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. In the case of heteroalkylene groups, the heteroatoms can also occupy one or both ends of the chain. Furthermore, in the case of alkylene and heteroalkylene linking groups, the orientation of the linking group is not implicitly indicated. In a selected embodiment, a heteroalkylene is a heteroalkyl group as defined above, in which two or more of the hydrogen atoms of the heteroalkyl group are replaced by bonds (i.e., this heteroalkylene may be trivalent). In some aspects, heteroalkyls or heteroalkylenes may be saturated. In some aspects, heteroalkylenes are unsubstituted. Optionally, heteroalkylenes may be substituted, for example, with one or more groups.

[0103] Unless otherwise specified, the term "halogen" generally means the elements of Group 7, preferably fluorine, chlorine, bromine, and iodine, more preferably fluorine, chlorine, and bromine, and even more preferably fluorine and chlorine.

[0104] Terms such as "substituted," "may be substituted," or "optionally substituted" generally mean, unless otherwise specified, that one or more hydrogen atoms may be independently substituted by substituents. Typical substituents include -X, -R, and -O. - -OR, -SR, -S - , -NR2, -NR3, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NRC(=O)R, -C(=O)R, -C(=O)NR2, -SO3 - , -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NR, -S(=O)R, -OP(=O)(OR) 2、 -P(=O)(OR)2, -PO4 3--PO3H2, -C(=O)R, -C(=O)X, -C(=S)R, -CO2R, -CO2, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR2, -C(=S)NR2, or -C(=NR)NR2, where each X is independently a halogen: -F, -Cl, -Br, or -I; each R is independently -H, -(C1~C 20 )alkyl (for example, -(C1~C 10 )alkyl or -(C1~C8)alkyl), -(C6~C 20 )aryl (for example, -(C6~C 10 )aryl or preferably C6-aryl), -(C3~C 14 ) Complex algebras (for example, -(C3~C 10 The substituent is a heterocycle or a -(C3-C8) heterocycle, a protecting group, or a prodrug moiety. Typical substituents also include (=O).

[0105] The term “aliphatic residue or aromatic residue,” as used herein, generally means an aliphatic substituent such as, for example, an alkyl residue, which may optionally be substituted by further aliphatic and / or aromatic substituents. As a non-limiting example, an aliphatic residue may be used for the direct linkage of such a molecule to the core structure (R 1 In this case, the residue can be a nucleic acid, enzyme, coenzyme, nucleotide, oligonucleotide, monosaccharide, polysaccharide, polymer, fluorophore, or optionally substituted benzene, as long as the linkage (for example, to an oxygen atom bonded to phosphorus) is aliphatic. Aromatic residues are substituents whose direct linkage to the core structure is part of an aromatic system, for example, optionally substituted phenyl, triazolyl, or pyridyl, or nucleotide, and in non-limiting examples, the direct linkage of a nucleotide to the core structure is, for example, via a phenyl residue. The term “aromatic residue” also includes heteroaromatic residues as used herein.

[0106] The term "peptide," unless otherwise specified, generally refers to an organic compound containing two or more amino acids covalently linked by peptide bonds (amide bonds). Peptides may be named based on the number of amino acids they contain; for example, a dipeptide contains two amino acid residues, a tripeptide contains three amino acids, and so on. Peptides containing 30 or fewer amino acids may be called oligopeptides, while those containing more than 30 amino acid residues may be called polypeptides. The amino acids and peptides according to this disclosure may also be modified with functional groups. Non-limiting examples include sugars, e.g., N-acetylgalactosamine (GalNAc), or protecting groups, e.g., fluorenyl methoxycarbonyl (Fmoc) modification or esterification.

[0107] Pharmaceutically acceptable salts This disclosure also relates to “pharmaceutically acceptable salts.” Any pharmaceutically acceptable salt may be used. In particular, the term “pharmaceutically acceptable salt” means a salt of the conjugate or compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. In particular, such salts may be low-toxicity inorganic or organic acid and base addition salts. Specifically, such salts include, but are not limited to, the following: (1) those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, or acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphor Acid addition salts formed with organic acids such as α-sulfonic acid, 4-methylbicyclo[2.2.2]-octo-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; or (2) salts formed when an acidic proton present in the parent compound is substituted with a metal ion, such as an alkali metal ion, an alkaline earth ion, or an aluminum ion, or when it coordinates with an organic base, such as ethanolamine, diethanolamine, triethanolamine, and N-methylglucamine. Salts further include, but are merely examples, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium; and if the compound contains a basic functional group, salts of non-toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate. In quaternary amines, counterions or anionic counterions may be used to maintain electronic neutrality.Examples of counterions include halide ions (e.g., F). - Cl - , Br - , I - ), NO3 - ClO4 - , OH - H2PO4 - HSO4 - These include sulfonate ions (e.g., methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphorsulfonate, naphthalene-2-sulfonate, and naphthalene-1-sulfonic acid-5-sulfonate), as well as carboxylate ions (e.g., acetate, ethanolate, propanoate, benzoate, glycerate, lactate, tartraate, and glycolate).

[0108] solvate As used herein, the term “solvate” may mean an aggregate comprising one or more molecules of the conjugate or compound described herein and one or more molecules of a solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the conjugates or compounds of this disclosure may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, and tetrahydrates, and as corresponding solvated forms. The compounds of the present invention may be true solvates, but in other cases, the compounds of the present invention may only retain incidental water, or may be a mixture of water and some incidental solvent.

[0109] In some aspects / modes, the present invention relates to an anti-TPBG antibody (e.g., its antibody-binding moiety) comprising an Fc-silencing mutation such as a leucine (L) to alanine (A) substitution (LALA mutation) at positions 234 and 235, and the anti-TPBG antibody is capable of binding to human trophoblast glycoprotein (TPBG) (e.g., having UniProt accession number: Q13641 or SEQ ID NO: 1); the anti-TPBG antibody is capable of cross-reactivity with marmoset (e.g., common marmoset) TPBG (e.g., having UniProtKB accession number: F7I0T3 or SEQ ID NO: 2) that has white tufts of hair around its ears; and the anti-TPBG antibody is capable of internal transfer, preferably by antigen-mediated internal transfer of the antibody.

[0110] In some aspects / embodiments, the present invention relates to a monoclonal human or humanized IgG1 anti-TPBG antibody, preferably comprising a kappa (κ) light chain.

[0111] In some aspects / applications, the present invention relates to a hybridoma that produces the antibody of the present invention.

[0112] In some aspects / applications, the present invention relates to nucleic acids encoding the antibodies of the present invention.

[0113] In some aspects / modes, the present invention relates to an expression vector comprising at least one nucleic acid molecule of the present invention.

[0114] The present invention further relates. In some aspects / portrayals, the present invention relates to isolated host cells (e.g., isolated recombinant host cells) comprising the vector and / or nucleic acids of the present invention.

[0115] In some aspects / modes, the present invention relates to an antibody-drug conjugate (ADC) comprising the anti-TPBG antibody of the present invention.

[0116] In some aspects / applications, the present invention relates to antibody-drug conjugates (ADCs) of the present invention, comprising an anti-TPBG antibody of the present invention (e.g., a humanized monoclonal TPBG-specific IgG1 antibody) conjugated to a cytotoxic payload; wherein (a) the cytotoxic payload is selected from the group consisting of camptothecin, mytansinoids, calicheamycin, duochamycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof; and / or (b) the cytotoxic payload is exatecan, DXD, SN38, camptothecin, topotecan, irinotecan (c) the cytotoxic payload is a camptothecin moiety C selected from the group consisting of berotecan, lulutotecan, rubitecan, silatecan, cocitecan, and gimatecan; and / or (c) the cytotoxic payload is conjugated via a cleavable linker (L), preferably the linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction, and more preferably the linker is cleavable by a protease, preferably a cathepsin such as cathepsin B; and / or (d) the linker (L) comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) the cytotoxic payload is exatecan conjugated via a chemical valine-citrulline-PAB free unit or a valine-alanine-PAB free unit, the free unit being cleavable by a protease.Preferably, the drug-to-antibody ratio (DAR) is in the range of 0 to 20, more preferably in the range of 4 to 8, and most preferably 4 or 8.

[0117] In some aspects / modes, the present invention relates to formula (I): Regarding antibody-drug conjugates (ADCs) having TIFF2026513098000002.tif34128 or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, Ab is the anti-TPBG antibody described herein; TIFF2026513098000003.tif6128 is either a double bond or TIFF2026513098000004.tif6128 is a single bond; If TIFF2026513098000005.tif6128 has a double bond, then V does not exist, or If TIFF2026513098000006.tif6128 is a single bond, then V is either H or (C1-C8) alkyl; If TIFF2026513098000007.tif6128 has a double bond, then X is R3-C, or If TIFF2026513098000008.tif6128 is a single join, then X is: The filename is TIFF2026513098000009.tif9128; Y is NR 5 , S, O, or CR 6 R 7 and; R 1 is an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 3is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 4 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 5 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 6 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 7 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; CU stands for connector unit; CM is the cytotoxic part; m is an integer in the range of 1 to 10; and n is an integer in the range of 1 to 20.

[0118] Preferably, R 3 is H or (C1-C8) alkyl, more preferably R 3 is H. Preferably, R 4 If present, is H or (C1-C8) alkyl; more preferably R 4 If present, it is H. Preferably, R 5 If present, is H or (C1-C8) alkyl; more preferably R 5 If present, it is H. Preferably, R 6 If present, is H or (C1-C8) alkyl; more preferably R 6 If present, it is H. Preferably, R 7 If present, is H or (C1-C8) alkyl; more preferably R 7 If it exists, it is H.

[0119] Preferably, TIFF2026513098000010.tif6128 has a double bond; V is absent; X is R3-C; and R 3 is H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 3 is H or (C1-C8) alkyl; more preferably, R 3 H is H.

[0120] more, TIFF2026513098000011.tif6128 represents a double bond; V is absent; X represents R3-C, and R3 represents H or (C1~C8) alkyl. Preferably, R 3 R3 represents H or (C1-C6) alkyl, more preferably H or (C1-C4) alkyl, and even more preferably H or (C1-C2) alkyl. In a preferred embodiment, R3 is H.

[0121] In some embodiments, TIFF2026513098000012.tif6128 may be a single bond; V is H or (C1~C8) alkyl, preferably V is H; X is TIFF2026513098000013.tif9128; R3 is H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; more preferably, R 3 is H or (C1-C8) alkyl, more preferably R 3 H is R 4 is H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 4 is H or (C1-C8) alkyl, preferably R 4 H is H.

[0122] In some embodiments, TIFF2026513098000014.tif6128 may represent a single bond; V may be H or (C1~C8) alkyl; X is R3 and R4 may represent TIFF2026513098000015.tif9128; and R3 and R4 may independently represent H or (C1-C8) alkyl. Preferably, R3 and R4 independently represent H or (C1-C6) alkyl, more preferably H or (C1-C4) alkyl, and even more preferably H or (C1-C2) alkyl. Preferably, R3 and R4 are the same; even more preferably R 3、 R 4、 R3 and V are the same. More preferably, R3 and R4 are both H. Preferably, V is H or (C1-C6) alkyl, more preferably H or (C1-C4) alkyl, and even more preferably H or (C1-C2) alkyl. Even more preferably, V is H. In a preferred embodiment, R3, R4, and V are each H.

[0123] The integer m is in the range of 1 to 10. Therefore, the integer m may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the integer m is in the range of 1 to 4. More preferably, the integer m is 1 or 2. Even more preferably, the integer m is 1.

[0124] The integer n is in the range of 1 to 20. Therefore, the integer n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Preferably, the integer n is in the range of 1 to 10. More preferably, the integer n is in the range of 2 to 10. Even more preferably, the integer n is in the range of 4 to 10. Even more preferably, the integer n is in the range of 6 to 10. Even more preferably, the integer n is 6, 7, 8, 9, or 10. Even more preferably, the integer n is in the range of 7 to 10. Even more preferably, the integer n is 7, 8, or 9. Even more preferably, the integer n is 7 or 8. Even more preferably, the integer n is 8.

[0125] The integer n is in the range of 1 to 20. Preferably, the integer n is in the range of 1 to 10. More preferably, the integer n is in the range of 2 to 8. Even more preferably, the integer n is 2, 3, 4, 5, or 6. Even more preferably, the integer n is in the range of 3 to 6. Even more preferably, the integer n is 3, 4, or 5. Even more preferably, the integer n is 4 or 5. Even more preferably, the integer n is 4.

[0126] Preferably, m is an integer in the range of 1 to 4, more preferably 1 or 2, and even more preferably 1; preferably, n is an integer in the range of 1 to 20, more preferably 1 to 10, even more preferably 2 to 10, even more preferably 4 to 10, even more preferably 6 to 10, even more preferably n is 6, 7, 8, 9, or 10, even more preferably n is in the range of 7 to 10, even more preferably n is 7, 8, or 9, even more preferably n is 7 or 8, and even more preferably n is 8.

[0127] Preferably, m is an integer in the range of 1 to 4, more preferably 1 or 2, and more preferably 1; preferably, n is an integer in the range of 1 to 20, more preferably 1 to 10, even more preferably 2 to 8; even more preferably, n is 2, 3, 4, 5, or 6; even more preferably, n is in the range of 3 to 6; even more preferably, n is 3, 4, or 5; even more preferably, n is 4 or 5, and even more preferably, n is 4.

[0128] Preferably, m is 1; preferably, n is an integer in the range of 1 to 20, more preferably 1 to 10, even more preferably 2 to 10, even more preferably 4 to 10, even more preferably 6 to 10, even more preferably n is 6, 7, 8, 9, or 10, even more preferably n is in the range of 7 to 10, even more preferably n is 7, 8, or 9, even more preferably n is 7 or 8, and even more preferably n is 8. Therefore, preferably, m is 1 and n is an integer in the range of 1 to 20. More preferably, m is 1 and n is an integer in the range of 1 to 10. Even more preferably, m is 1 and n is an integer in the range of 2 to 10. Even more preferably, m is 1 and n is an integer in the range of 4 to 10. Even more preferably, m is 1 and n is an integer in the range of 6 to 10. More preferably, m is 1 and n is 6, 7, 8, 9, or 10. More preferably, m is 1 and n is an integer in the range of 7 to 10. More preferably, m is 1 and n is 7, 8, or 9. More preferably, m is 1 and n is 7 or 8. Even more preferably, m is 1 and n is 8.

[0129] Preferably, m is 1; preferably, n is an integer in the range of 1 to 20, more preferably 1 to 10, even more preferably 2 to 8; even more preferably, n is 2, 3, 4, 5, or 6, even more preferably n is in the range of 3 to 6; even more preferably n is 3, 4, or 5; even more preferably n is 4 or 5, and even more preferably n is 4. Therefore, preferably, m is 1 and n is an integer in the range of 1 to 20. More preferably, m is 1 and n is an integer in the range of 1 to 10. Even more preferably, m is 1 and n is an integer in the range of 2 to 8. Even more preferably, m is 1 and n is 2, 3, 4, 5, or 6. Even more preferably, m is 1 and n is in the range of 3 to 6. Even more preferably, m is 1 and n is 3, 4, or 5. More preferably, m is 1 and n is 4 or 5. Even more preferably, m is 1 and n is 4.

[0130] In some embodiments, the number of cytotoxic moieties (CMs) per antibody Ab may be 1 to 20. Preferably, the number of cytotoxic moieties (CMs) per antibody Ab is 1 to 14. More preferably, the number of cytotoxic moieties (CMs) per antibody Ab is 2 to 14. More preferably, the number of cytotoxic moieties (CMs) per antibody Ab is 4 to 14. Even more preferably, the number of cytotoxic moieties (CMs) per antibody Ab is 5 to 12. Even more preferably, the number of cytotoxic moieties (CMs) per antibody Ab is 6 to 12. Even more preferably, the number of cytotoxic moieties (CMs) per antibody Ab is 7 to 10. Even more preferably, the number of cytotoxic moieties (CMs) per antibody Ab is 8.

[0131] In some embodiments, the number of cytotoxic moieties (CMs) per antibody Ab may be 1 to 20. Preferably, the number of cytotoxic moieties per antibody Ab is 1 to 14. More preferably, the number of cytotoxic moieties per antibody Ab is 1 to 12. More preferably, the number of cytotoxic moieties per antibody Ab is 2 to 10. Even more preferably, the number of cytotoxic moieties per antibody Ab is 2 to 8. Even more preferably, the number of cytotoxic moieties per antibody Ab is 2 to 6. Even more preferably, the number of cytotoxic moieties per antibody Ab is 3 to 5. Even more preferably, the number of cytotoxic moieties per antibody Ab is 4.

[0132] Ab is an anti-TPBG antibody as described herein. Any anti-TPBG antibody as described herein may be used.

[0133] Base Y Base Y is NR 5 , S, O, and CR 6 R 7 Selected from the group consisting of R. 5 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 5 is H or (C1-C8) alkyl; more preferably, R 5 H is R 6 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 6 is H or (C1-C8) alkyl; more preferably, R 6 H is R 7 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 7 is H or (C1-C8) alkyl, more preferably R 7 H is H.

[0134] Preferably, Y is selected from the group consisting of NH, S, O, and CH2. More preferably, Y is NH, S, or O. In some embodiments, Y is CH2. In some embodiments, Y is O. In some embodiments, Y is S.

[0135] In a very preferred embodiment, Y is NH.

[0136] Group R 1 R 1 is an optionally substituted aliphatic residue or an optionally substituted aromatic residue.

[0137] R 1 may represent an optionally substituted (C1-C8) alkyl.

[0138] R 1 may represent a (C1-C8) alkyl optionally substituted by at least one of F, Cl, Br, I, -NO2, -N((C1-C8) alkyl)H, -NH2, -N3, -N((C1-C8) alkyl)2, =O, (C3-C8) cycloalkyl, -S-S-((C1-C8) alkyl), (C2-C8) alkenyl, or (C2-C8) alkynyl.

[0139] R 1 may represent an optionally substituted phenyl.

[0140] R 1 may represent a phenyl optionally independently substituted by at least one of (C1-C8) alkyl, F, Cl, I, Br, -NO2, -N((C1-C8) alkyl)H, -NH2, or -N((C1-C8) alkyl)2.

[0141] R 1 may represent an optionally substituted 5- or 6-membered aromatic heterocycle, such as pyridyl.

[0142] R 1This may represent a (C1-C8) alkyl group, a (C1-C8) alkyl group substituted with -SS-(C1-C8) alkyl group, a (C1-C8) alkyl group substituted with an optionally substituted phenyl group, or a phenyl group, or a phenyl group substituted with -NO2.

[0143] R 1 may represent methyl, ethyl, propyl, or butyl, preferably methyl or ethyl, more preferably ethyl.

[0144] Polyethylene glycol units Preferably, R 1 R is a polyethylene glycol unit. In some embodiments, R 1 This is a polyethylene glycol unit comprising 1 to 100, preferably 2 to 50, more preferably 3 to 45, even more preferably 4 to 40, even more preferably 6 to 35, and still more preferably 8 to 30 ethylene glycol subunits, wherein each ethylene glycol subunit has the following structure: It has TIFF2026513098000016.tif9128. Throughout this specification, the structure: TIFF2026513098000017.tif9128 is represented as an "ethylene glycol subunit".

[0145] Preferably, R 1 This is a polyethylene glycol unit comprising 16 to 30, more preferably 20 to 28, even more preferably 22, 23, 24, 25, or 26, and even more preferably 23, 24, or 25 ethylene glycol subunits, each of which has the following structure: It has TIFF2026513098000018.tif9128. In a preferred embodiment, R 1 This is a polyethylene glycol unit containing 24 or approximately 24 ethylene glycol subunits, each of which has the following structure: It has TIFF2026513098000019.tif9128.

[0146] More preferably, R 1 is of the structure: It is a polyethylene glycol unit having TIFF2026513098000020.tif15128, wherein, TIFF2026513098000021.tif10128 indicates the position of O bonded to phosphorus; K F is H (hydrogen) or a cap group described herein; preferably, K F is -H (hydrogen), -PO3H, -(C1~C 10 ) alkyl, -(C1~C 10 ) alkyl-SO3H, -(C2~C 10 ) alkyl-CO2H, -(C2~C 10 ) alkyl-OH, -(C2~C 10 ) alkyl-NH2, -(C2~C 10 ) alkyl-NH(C1~C3) alkyl, and -(C2~C 10 ) alkyl-N((C1~C3) alkyl)2 selected from the group consisting of; more preferably, K F is H; and o is an integer in the range of 1 to 100.

[0147] The integer o is the repeating unit in the polyethylene glycol unit The numbers in TIFF2026513098000022.tif12128 are shown. The integer o may be in the range of 1 to 100. Preferably, o is in the range of 2 to 50. More preferably, o is in the range of 3 to 45. Even more preferably, o is in the range of 4 to 40. Even more preferably, o is in the range of 6 to 35. Even more preferably, o is in the range of 8 to 30. In some embodiments, o is 12 or about 12. Even more preferably, o is in the range of 16 to 30. Even more preferably, o is in the range of 20 to 28. Even more preferably, o is 22, 23, 24, 25, or 26. Even more preferably, o is 23, 24, or 25. In preferred embodiments, o is 24 or about 24.

[0148] Typically, polydisperse polyethylene glycol, monodisperse polyethylene glycol, and discrete polyethylene glycol can be used as polyethylene glycol units. Polydisperse polyethylene glycol is a heterogeneous mixture of size and molecular weight, while monodisperse polyethylene glycol is typically purified from a heterogeneous mixture and therefore yields a single chain length and molecular weight. The preferred polyethylene glycol unit is discrete polyethylene glycol, i.e., a compound synthesized stepwise rather than through a polymerization process. Discrete polyethylene glycol provides a single molecule with a defined and specified chain length.

[0149] The polyethylene glycol units provided herein comprise one or more polyethylene glycol chains. The polyethylene glycol chains may be linked together, for example, in a linear, branched, or star-shaped configuration. Optionally, at least one of the polyethylene glycol chains may be derivatized at one end for covalent bonding to an oxygen atom bonded to phosphorus.

[0150] A polyethylene glycol unit is bound to an antibody-drug conjugate (or intermediate thereof) at the oxygen atom bonded to phosphorus. The other end (or more ends) of the polyethylene glycol unit are unbound in a free state and may take the form of hydrogen, methoxy, carboxylic acid, alcohol, or other suitable functional group, e.g., any cap group as described herein. The methoxy, carboxylic acid, alcohol, or other suitable functional group acts as a cap for the terminal polyethylene glycol subunit of the polyethylene glycol unit. "Unbound" means that the polyethylene glycol unit is not bound at its unbound site to a cytotoxic moiety (CM), a receptor binding site, or a component of a connector unit (CU) that connects the cytotoxic moiety and / or the antibody (Ab). If the polyethylene glycol unit comprises multiple polyethylene glycol chains, these multiple polyethylene glycol chains may be the same or different chemical moieties (e.g., polyethylene glycols with different molecular weights or numbers of subunits). These multiple polyethylene glycol chains are bound at a single binding site to the oxygen atom bonded to phosphorus. Those skilled in the art will understand that, in addition to comprising repeating polyethylene glycol subunits, polyethylene glycol units may also contain non-polyethylene glycol material (for example, to facilitate the interconnection of multiple polyethylene glycol chains or to facilitate the linkage to the phosphorus-bonded oxygen atom). Non-polyethylene glycol material means atoms in the polyethylene glycol unit that are not part of the repeating -CH2CH2O- subunits. In embodiments provided herein, a polyethylene glycol unit may include two monomeric polyethylene glycol chains interconnected via non-polyethylene glycol elements. In other embodiments provided herein, a polyethylene glycol unit may include two linear polyethylene glycol chains bonded to a central core bonded to the phosphorus-bonded oxygen atom (i.e., the polyethylene glycol unit is branched).

[0151] Several methods for polyethylene glycol bonding are available to those skilled in the art [e.g., EP 0 401 384 (PEG linkage to G-CSF); U.S. Patent No. 5,757,078 (PEGylation of EPO peptides); U.S. Patent No. 5,672,662 (polyethylene glycol and related polymers mono substituted with propionic or butanoic acids and functional derivatives thereof for biotechnical applications); U.S. Patent No. 6,077,939 (PEGylation of the N-terminal α-carbon of peptides); and Veronese (2001) Biomaterials 22:405-417 (Review on PEGylation of peptides and proteins)].

[0152] In preferred embodiments, the polyethylene glycol unit is directly bonded to the oxygen atom bonded to phosphorus. In these embodiments, the polyethylene glycol unit does not contain a functional group for bonding to the oxygen atom bonded to phosphorus; that is, the oxygen atom is directly bonded to the carbon atom of the polyethylene glycol unit, preferably to the CH2 of the polyethylene glycol unit.

[0153] In one group of embodiments, a polyethylene glycol unit comprises at least one ethylene glycol subunit, preferably at least two ethylene glycol subunits, more preferably at least three ethylene glycol subunits, even more preferably at least four ethylene glycol subunits, even more preferably at least six ethylene glycol subunits, and even more preferably at least eight ethylene glycol subunits. In some such embodiments, a polyethylene glycol unit comprises about 100 or fewer ethylene glycol subunits, preferably about 50 or fewer ethylene glycol subunits, more preferably about 45 or fewer ethylene glycol subunits, more preferably about 40 or fewer ethylene glycol subunits, more preferably about 35 or fewer ethylene glycol subunits, and even more preferably about 30 or fewer ethylene glycol subunits.

[0154] In one group of embodiments, a polyethylene glycol unit comprises one or more linear polyethylene glycol chains, each of which has at least one ethylene glycol subunit, preferably at least two ethylene glycol subunits, more preferably at least three ethylene glycol subunits, even more preferably at least four ethylene glycol subunits, even more preferably at least six ethylene glycol subunits, and still more preferably at least eight ethylene glycol subunits. In a preferred embodiment, a polyethylene glycol unit comprises, in total, at least one ethylene glycol subunit, preferably at least two ethylene glycol subunits, more preferably at least three, even more preferably at least four, even more preferably at least six, or even more preferably at least eight ethylene glycol subunits. In some such embodiments, a polyethylene glycol unit comprises, in total, about 100 or fewer ethylene glycol subunits, preferably about 50 or fewer ethylene glycol subunits, more preferably about 45 or fewer ethylene glycol subunits, even more preferably about 40 or fewer ethylene glycol subunits, even more preferably about 35 or fewer ethylene glycol subunits, and still more preferably about 30 or fewer ethylene glycol subunits.

[0155] In another group of embodiments, the polyethylene glycol unit comprises a total of 1 to 100, preferably 2 to 50, more preferably 3 to 45, even more preferably 4 to 40, even more preferably 6 to 35, and still more preferably 8 to 30 ethylene glycol subunits. In any one of these embodiments, the ethylene glycol subunits may be any ethylene glycol subunits described herein.

[0156] In another group of embodiments, the polyethylene glycol unit comprises one or more linear polyethylene glycol chains having a total of 1 to 100, preferably 2 to 50, more preferably 3 to 45, even more preferably 4 to 40, even more preferably 6 to 35, and still more preferably 8 to 30 ethylene glycol subunits.

[0157] In another group of embodiments, the polyethylene glycol unit is a linear single polyethylene glycol chain having at least one ethylene glycol subunit, preferably at least two ethylene glycol subunits, more preferably at least three ethylene glycol subunits, even more preferably at least six ethylene glycol subunits, and even more preferably at least eight ethylene glycol subunits. Optionally, in any one of these embodiments, the linear single polyalkylene glycol chain may be derivatized.

[0158] In another group of embodiments, the polyethylene glycol unit is a linear single polyethylene glycol chain having 1 to 100, preferably 2 to 50, more preferably 3 to 45, more preferably 4 to 40, more preferably 6 to 35, and more preferably 8 to 30 ethylene glycol subunits. Optionally, in any one of these embodiments, the linear single polyethylene glycol chain may be derivatized.

[0159] In any one of the embodiments provided herein, R 1 Exemplary linear polyethylene glycol units that can be used are as follows: In formula TIFF2026513098000023.tif40128, the wavy lines indicate the bonding sites to the oxygen atom bonded to phosphorus; R 20 is a PEG-linked unit; preferably, R 20 It does not exist; R 21 R is a PEG capping unit (as specified herein).21 is "K F It is also written as ""; R 22 This is a PEG linking unit (i.e., a unit for linking multiple PEG subunit chains together); n is independently selected from 1 to 100, preferably 2 to 50, more preferably 3 to 45, more preferably 4 to 40, even more preferably 6 to 35, and still more preferably 8 to 30; e is between 2 and 5; Each n' is independently selected from 1 to 100, preferably 2 to 50, more preferably 3 to 45, more preferably 4 to 40, even more preferably 6 to 35, and even more preferably 8 to 30. In a preferred embodiment, the polyethylene glycol unit contains at least 1, preferably at least 2, more preferably at least 3, more preferably at least 4, more preferably at least 6, and even more preferably at least 8 ethylene glycol subunits. In some embodiments, the polyethylene glycol unit contains 100 or fewer, preferably 50 or fewer, more preferably 45 or fewer, more preferably 40 or fewer, more preferably 35 or fewer, and even more preferably about 30 or fewer ethylene glycol subunits. 20 If it is not present, the (CH2CH2O) subunit is directly bonded to the oxygen atom bonded to phosphorus.

[0160] Preferably, linear polyethylene glycol units are The formula is TIFF2026513098000024.tif9128, where the wavy line indicates the bonding site to the oxygen atom bonded to phosphorus; R 20 , R 21 (In this specification, "K F (Also written as ") and n are as defined herein; more preferably, R 20 It does not exist. In a preferred embodiment, n is 12 or about 12. In a preferred embodiment, n is 24 or about 24. Preferably, R 21 H is H.

[0161] Polyethylene glycol bond unit R 20 If present, it is a part of a polyethylene glycol unit and plays the role of linking the polyethylene glycol unit to the oxygen atom bonded to phosphorus. In this regard, the oxygen atom bonded to phosphorus forms a bond with the polyethylene glycol unit. In an exemplary embodiment, the PEG bond unit R 20 If it exists, * -(C1~C 10 ) Alkyl- # , * -Ariren- # , * -(C1~C 10 )alkyl-O- # , * -(C1~C 10 )alkyl-C(O)- # , * -(C1~C 10 )alkyl-C(O)O- # , * -(C1~C 10 )alkyl-NH- # , * -(C1~C 10 ) Alkyl-S- # , * -(C1~C 10 )alkyl-C(O)-NH- # , * -(C1~C 10 )alkyl-NH-C(O)- # , and * -CH2-CH2SO2-(C1~C 10 ) Alkyl- # Selected from the group consisting of; where, * The symbol indicates the bond point to the oxygen atom bonded to phosphorus, and the symbol # indicates the bond point to the ethylene glycol unit.

[0162] PEG linked unit R 22 R is a non-PEG substance that, if present, is part of a polyethylene glycol unit and plays a role in linking two or more chains of repeating -CH2CH2O- subunits. In an exemplary embodiment, the PEG linking unit R22 If it exists, * -(C1~C 10 )alkyl-C(O)-NH- # , * -(C1~C 10 )alkyl-NH-C(O)- # , * -(C2~C 10 )alkyl-NH- # , * -(C2~C 10 )alkyl-O- # , * -(C1~C 10 ) Alkyl-S- # ,or * -(C2~C 10 )alkyl-NH- # A group consisting of these elements is independently selected; here, * The symbol indicates the bond point to the oxygen atom of an ethylene glycol subunit, and the symbol # indicates the bond point to the carbon atom of another ethylene glycol subunit.

[0163] R 21 The base is referred to as "K" in this specification. F It is also written as " and in exemplary embodiments it may be H (hydrogen) or a cap group as described herein. Preferably R 21 -H, -PO3H, -(C1~C 10 )alkyl, -(C1~C 10 )alkyl-SO3H,-(C2~C 10 )alkyl-CO2H,-(C2~C 10 )alkyl-OH, -(C2~C 10 )alkyl-NH2,-(C2~C 10 )alkyl-NH(C1~C3)alkyl, and -(C2~C 10 ) Independently selected from the group consisting of alkyl-N((C1~C3)alkyl)2. In some embodiments, R 21 is, -(C1~C 10 ) Alkyl, especially methyl. More preferably, R 21 H is H.

[0164] In any one of the embodiments provided herein, R 1 Examples of linear polyethylene glycol units that can be used are as follows: The formula is TIFF2026513098000025.tif57128, where the dashed line indicates the bonding site to the oxygen atom bonded to phosphorus; each n is 1 to 100, preferably 2 to 50, more preferably 3 to 45, even more preferably 4 to 40, even more preferably 6 to 35, and still more preferably 8 to 30. In some embodiments, n is about 12. In some embodiments, n is about 24.

[0165] In some embodiments, polyethylene glycol units are approximately 300 daltons to approximately 5 kilodaltons, approximately 300 daltons to approximately 4 kilodaltons, approximately 300 daltons to approximately 3 kilodaltons, approximately 300 daltons to approximately 2 kilodaltons, or approximately 300 daltons to approximately 1 kilodalton. In some such aspects, polyethylene glycol units may have at least 6 ethylene glycol subunits or at least 8 ethylene glycol subunits. In some such aspects, polyethylene glycol units may have at least 6 ethylene glycol subunits or at least 8 ethylene glycol subunits, provided that the number of ethylene glycol subunits is 100 or less, preferably 50 or less. In some embodiments, polyethylene glycol units are approximately 300 daltons to approximately 5 kilodaltons, approximately 300 daltons to approximately 4 kilodaltons, approximately 300 daltons to approximately 3 kilodaltons, approximately 300 daltons to approximately 2 kilodaltons, or approximately 300 daltons to approximately 1 kilodalton. In some such aspects, the polyethylene glycol unit may have at least six ethylene glycol subunits or at least eight ethylene glycol subunits. In some aspects, the polyethylene glycol unit may have at least six ethylene glycol subunits or at least eight ethylene glycol subunits, provided that the number of ethylene glycol subunits is 100 or less, preferably 50 or less.

[0166] In some embodiments, R 1 If is a polyethylene glycol unit, there are no other ethylene glycol subunits and / or alkylene glycol subunits present in the antibody-drug conjugate of formula (I) (i.e., there are no ethylene glycol subunits and / or alkylene glycol subunits in any of the other components of the antibody-drug conjugate, for example, in the connector units (CUs) provided herein). In other aspects, R 1If is a polyethylene glycol unit, then the conjugate of formula (I) contains 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer other ethylene glycol subunits and / or alkylene glycol subunits (i.e., among the other components of the conjugate, for example, among the connector units (CUs) provided herein, there are 8, 7, 6, 5, 4, 3, 2, or 1 or fewer other ethylene glycol subunits and / or alkylene glycol subunits).

[0167] When referring to ethylene glycol subunits and / or alkylene glycol subunits, it will be recognized that, depending on the context, for example, when referring to a group of conjugates or intermediate compounds, and when using polydisperse polyethylene glycol, the number of subunits may represent the average number.

[0168] "CU": Connector unit This disclosure provides an antibody-drug conjugate (ADC) in which an anti-TPBG antibody (Ab), as described herein, is linked to a cytotoxic moiety (CM). According to this disclosure, the antibody (Ab) can be linked to a cytotoxic moiety via covalent bonding of a group Y and a connector unit CU. As used herein, the "connector unit" CU is any chemical moiety to which a group Y, e.g., NH, can be linked to another moiety, e.g., a cytotoxic moiety. In particular, the connector unit CU forms part of a linker (L) that can link the antibody of the present invention to one or more cytotoxic moieties CM (or drug moieties or cytotoxic payloads), as described herein. In this regard, formula (I) as described herein: TIFF2026513098000026.tif34128 is mentioned again. Thus, the cytotoxic portion CM can be linked to Y via the connector unit CU. In equation (I), Ab, TIFF2026513098000027.tif6128, V, X, Y, R 1CU, CM, m, and n are as defined herein. The connector unit CU plays a role in connecting Y to the cytotoxic moiety (CM). The connector unit CU is any chemical moiety that can link Y to the cytotoxic moiety CM. Specifically, the connector unit CU links Y to the cytotoxic moiety CM via covalent bonding. The connector unit is a bifunctional or polyfunctional moiety that can be used to link the cytotoxic moiety CM and Y to form an antibody-drug conjugate of formula (I). The terms “connector unit,” “connector reagent,” “linker reagent,” “crosslinking reagent,” “linker derived from crosslinking reagent,” and “linker” may be used synonymously throughout this disclosure.

[0169] Connector units (CUs) can be sensitive to cleavage such as enzymatic cleavage, acid-inducible cleavage, photo-inducible cleavage, and disulfide bond cleavage (cleavable connector units). Enzymatic cleavage preferably includes, but is not limited to, protease-inducible, peptidase-inducible, esterase-inducible, glycosidase-inducible, phosphatase-inducible, and sulfatase-inducible cleavage, under conditions in which the cytotoxic moiety and / or antibody (Ab) remain active. Alternatively, connector units can be substantially resistant to cleavage (e.g., stable connector units or uncleavable connector units). In some aspects, connector units (CUs) may be procharged connector units, hydrophilic connector units, PEG-based connector units, or dicarboxylic acid-based connector units. Accordingly, in some embodiments of any one of the antibody-drug conjugates disclosed herein, the connector unit (CU) is selected from the group consisting of cleavable connector units, non-cleavable connector units, hydrophilic connector units, PEG-based connector units, procharged connector units, peptide-based connector units, and dicarboxylic acid-based connector units. Preferably, the connector unit CU is a cleavable connector unit. In some embodiments, the connector unit CU is a non-cleavable connector unit.

[0170] Preferably, as described herein, the connector unit CU is cleavable. In some embodiments, CU is a connector unit sensitive to enzymatic cleavage. In some embodiments, CU is an acid-unstable connector unit, a photo-unstable connector unit, a peptidase-cleavable connector unit, a protease-cleavable connector unit, an esterase-cleavable connector unit, a glycosidase-cleavable connector unit, a phosphatase-cleavable connector unit, a sulfatase-cleavable connector unit, a disulfide bond-reducing connector unit, a hydrophilic connector unit, a procharged connector unit, a PEG-based connector unit, or a dicarboxylic acid-based connector unit. Preferably, the connector unit CU is cleavable by proteases, glucuronidases, sulfatases, phosphatases, esterases, or by disulfide reduction. Preferably, the connector unit is a peptidase-cleavable connector unit. Other preferred connector units are cleavable by proteases.

[0171] An uncleavable connector unit is any chemical moiety that can stable covalently link (or connect) to the cytotoxic moiety Y and does not belong to the category of cleavable connector units listed herein. Therefore, an uncleavable connector unit is substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, protease-induced cleavage, glycosidase-induced cleavage, phosphatase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Furthermore, “uncleavable” means the ability of a chemical bond in the connector unit or its attachment to the connector unit to resist cleavage induced by acid, photo-unstable cleavage agents, peptidases, proteases, glycosidases, phosphatases, esterases, or chemical or physiological compounds that cleave disulfide bonds, provided that the cytotoxic moiety or antibody (Ab) does not lose its activity.

[0172] Acid-unstable connector units are those that can be cleaved at an acidic pH. For example, certain intracellular compartments such as endosomes and lysosomes have an acidic pH (pH 4-5) and provide suitable conditions for cleaving acid-unstable connector units.

[0173] Some connector units can be cleaved by peptidases, i.e., they are peptidase-cleavable connector units. In this regard, certain peptides are readily cleaved inside or outside the cell. See, for example, Trout et al., 79 Proc. Natl. Acad. Sci. USA, 626-629 (1982) and Umemoto et al. 43 Int. J. Cancer, 677-684 (1989). Peptides consist of α-amino acids and peptide bonds, which are chemically amide bonds between the carboxylate of the first amino acid and the amino group of the second amino acid.

[0174] Some connector units (CU) can be cleaved by esterases, i.e., they are esterase-cleavable connector units. In this regard, certain esters can be cleaved by esterases present inside or outside the cell. Esters are formed by the condensation of carboxylic acids and alcohols. Simple esters are those produced by the reaction of simple alcohols, such as aliphatic alcohols, as well as small cyclic alcohols and small aromatic alcohols.

[0175] Procharged connector units are derived from charged crosslinking reagents that retain their charge after being incorporated into antibody-drug conjugates. Examples of procharged connector units (or linkers) can be found in US 2009 / 0274713.

[0176] Preferably, the connector unit CU is cleavable, as described herein. Exemplary examples include the connector unit being cleavable by proteases, glucuronidases, sulfatases, phosphatases, esterases, or by disulfide reduction. Preferably, the connector unit CU is cleavable by proteases. More preferably, the connector unit is cleavable by cathepsins, such as cathepsin B in particular. The connector unit may include a dipeptide moiety, such as a valine-citrulline moiety or a valine-alanine moiety, which can be cleaved by a cathepsin such as cathepsin B. Thus, in some embodiments, the connector unit includes a valine-citrulline moiety. In some embodiments, the connector unit includes a valine-alanine moiety. The connector unit may include cleavage sites. The term "cleavage site" may mean a chemical portion that is recognized by an enzyme and subsequently cleaved, for example, by hydrolysis. As an exemplary example, the cleavage site is a sequence of amino acids that is recognized by a protease or peptidase and hydrolyzed by the protease or peptidase. In some embodiments, the cleavage site is a dipeptide. In some embodiments, the cleavage site is a valine-citrulline moiety. In some embodiments, the cleavage site is a valine-alanine moiety.

[0177] Second spacer unit In a preferred embodiment, the connector unit (CU) includes a second spacer unit -A- bonded to -Y-. The second spacer unit plays a role in connecting -Y- to another part of the connector unit, or to a cytotoxic moiety (-CM), if present. As will be readily understood by those skilled in the art, this depends on whether or not another part of the connector unit is present. The second spacer unit (-A-) may be any chemical group or chemical moiety that can connect -Y- to another part of the connector unit, or to a cytotoxic moiety (-CM), if present, depending on whether or not another part of the connector unit is present. In this regard, -Y- is bonded to the second spacer unit (-A-) as described herein. The second spacer unit (-A-) may include, or may be, a functional group that can form a bond to another part of the connector unit or to a cytotoxic moiety (-CM), if present. Again, this also depends on whether or not another part of the connector is present. Preferably, the functional group that can form a bond to another part of the connector unit or to the cytotoxic moiety (-CM) is, for example, It is a carbonyl group, represented as TIFF2026513098000028.tif12128 or -C(O)-.

[0178] The second spacer unit may be any spacer known to those skilled in the art, for example, a linear or branched hydrocarbon-based portion. The second spacer unit may also include, but is not limited to, a cyclic portion, such as an aromatic portion. If the second spacer unit is a hydrocarbon-based portion, the main chain of the second spacer unit may contain only carbon atoms, but may also contain heteroatoms such as oxygen (O) atoms, nitrogen (N) atoms, or sulfur (S) atoms, and / or may contain a carbonyl group (C=O). The second spacer unit may be, for example, (C1~C 20) may contain or be composed of carbon atom chains. In a typical embodiment for hydrocarbon-based second spacer units, the spacing portion contains 1 to about 150, 1 to about 100, 1 to about 75, 1 to about 50, or 1 to about 40, or 1 to about 30, or 1 to about 20 main chain atoms, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19. Those skilled in the art know how to select a suitable second spacer unit.

[0179] In some embodiments, if a second spacer unit (-A-) is present, * -(C1~C 10 ) Alkylene-C(O)- # , * -(C3~C8)Carbocyclo-C(O)- # , * -Arirene-C(O)- # , * -(C1~C 10 )alkylene-arirene-C(O)- # , * -Ariren- (C1~C 10 ) Alkylene-C(O)- # , * -(C1~C 10 )alkylene-(C3~C8)carbocyclo-C(O)- # , * -(C3~C8)Carbocyclo-(C1~C 10 ) Alkylene-C(O)- # , * -(C3~C8) Heterocyclo-C(O)- # , * -(C1~C 10 )alkylene-(C3~C8)heterocyclo-C(O)- # , and * -(C3~C8) heterocyclo-(C1~C 10 ) Alkylene-C(O)- # Selected from the group consisting of; *indicates a binding point to -Y-; # indicates a binding point to another part of the connector unit, if present, or to the cytotoxic portion (-CM), depending on whether another part of the connector unit is present. Preferably, if a second spacer unit (-A-) is present, * -(C3~C8)Carbocyclo-C(O)- # , * -Arirene-C(O)- # , and * -(C3~C8) Heterocyclo-C(O)- # Selected from the group consisting of; * indicates a binding point to -Y-; # indicates a binding point to another part of the connector unit, or to the cytotoxic part (-CM), depending on whether another part of the connector unit exists.

[0180] In another embodiment, if a second spacer unit (-A-) is present, * -(C1~C 10 ) Alkilen- # , * -(C3~C8) Carbocyclo- # , * -Ariren- # , * -(C1~C 10 )Alkilen-Arirene- # , * -Ariren- (C1~C 10 ) Alkilen- # , * -(C1~C 10 )alkylene-(C3~C8)carbocyclo- # , * -(C3~C8)Carbocyclo-(C1~C 10 ) Alkilen- # , * -(C3~C8) heterocyclo- # , * -(C1~C 10 )alkylene-(C3~C8)heterocyclo- # , and * -(C3~C8) heterocyclo-(C1~C 10 ) Alkilen- #They may be selected from the group consisting of; * indicates a binding point to -Y-; # indicates a binding point to another part of the connector unit, if present, or to the cytotoxic portion (-CM), depending on whether another part of the connector unit is present. Preferably, if a second spacer unit (-A-) is present, * -(C3~C8) Carbocyclo- # , * -Ariren- # , and * -(C3~C8) heterocyclo- # They may be selected from the group consisting of; * indicates a binding point to -Y-; # indicates a binding point to another part of the connector unit, or to the cytotoxic part (-C), depending on whether another part of the connector unit exists.

[0181] Preferably, the second spacer unit -A- is The filename is TIFF2026513098000029.tif26128, and in the formula, TIFF2026513098000030.tif20128 is a 5- or 6-membered cyclic carbon; * indicates a binding point to -Y-; # indicates a binding point to another part of the connector unit, if present, or to the cytotoxic part (-CM), depending on whether another part of the connector unit is present. The cyclic carbon may be aromatic or non-aromatic. Preferably, the second spacer unit -A- is TIFF2026513098000031.tif26128, and in the formula, TIFF2026513098000032.tif19128 is a 5-membered or 6-membered heteroring containing one, two, or three heteroatoms independently selected from the group consisting of N, O, and S; * indicates a binding site to -Y-; # indicates a binding site to another part of the connector unit, if present, or to the cytotoxic moiety (-CM), depending on whether another part of the connector unit is present. The heterocycle may be aromatic or aromatic.

[0182] more, TIFF2026513098000033.tif26128 is, Selected from the group consisting of TIFF2026513098000034.tif28128, where A, B, C, and D are each independently selected from N (nitrogen) and CH; preferably, at least one of A, B, C, and D is CH; more preferably, at least two of A, B, C, and D are CH; even more preferably, at least three of A, B, C, and D are CH, and even more preferably, each of A, B, C, and D is CH; * indicates a binding point to -Y-; # indicates a binding point to another part of the connector unit, or to the cytotoxic portion (-CM), depending on whether another part of the connector unit is present. More preferably, TIFF2026513098000035.tif26128 is, TIFF2026513098000036.tif28128, where A, B, C, and D are each independently selected from N (nitrogen) and CH; preferably, at least one of A, B, C, and D is CH; more preferably, at least two of A, B, C, and D are CH; even more preferably, at least three of A, B, C, and D are CH, and even more preferably, each of A, B, C, and D is CH; where, * indicates a binding point to -Y-; # indicates a binding point to another part of the connector unit, or to the cytotoxic portion (-CM), depending on whether another part of the connector unit is present. More preferably, TIFF2026513098000037.tif26128 is, TIFF2026513098000038.tif25128, where A, B, C, and D are each independently selected from N (nitrogen) and CH; preferably, at least one of A, B, C, and D is CH; more preferably, at least two of A, B, C, and D are CH; even more preferably, at least three of A, B, C, and D are CH, and even more preferably, each of A, B, C, and D is CH; where, * indicates a binding point to -Y-; # indicates a binding point to another part of the connector unit, if present, or to the cytotoxic portion (-CM), depending on whether another part of the connector unit is present. In a very preferred embodiment, the second spacer unit A is TIFF2026513098000039.tif25128, and in the formula, * indicates a binding point to -Y-; # indicates a binding point to another part of the connector unit, or to the cytotoxic part (-CM), depending on whether another part of the connector unit exists.

[0183] In another embodiment, the second spacer unit (-A-) is: TIFF2026513098000040.tif19128 may be; m and n are each an integer independently of, for example, 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 1, preferably m is 1 and n is 1; * The symbol indicates the position of -Y-, and the symbol # indicates the binding point to another part of the connector unit, or to the cytotoxic part (-CM), depending on whether another part of the connector unit exists. Such a second spacer unit is particularly indicated by an asterisk ( * The carbon adjacent to the ) may optionally be substituted with, for example, one or two (C1-C8) alkyl groups.

[0184] Connector unit * -A a -W w -B b -## In some embodiments, the connector unit CU is given by formula: * -A a -W w -B b - ## The formula comprises, where -A- is a second spacer unit as described herein; a is 0 or 1; each -W- is independently an amino acid; w is independently an integer in the range of 0 to 12; -B- is a first spacer unit; b is 0 or 1; * indicates a binding site to -Y-; ## indicates a binding site to the cytotoxic moiety CM. In this specification, the symbol "W" indicates a binding site to the cytotoxic moiety CM. w " or -"W w The combination of W and the accompanying integer w is also referred to as an "amino acid unit." Appropriate second spacer units, amino acid units, and first spacer units are described, for example, in WO 2004 / 010957 A2.

[0185] structure * -A a -W w -B b - ## In a connector unit having the second spacer unit, the amino acid unit -W w - plays the role of connecting -Y- to -A-. The second spacer unit (-A-) may be any second spacer unit as described herein. If present, the second spacer unit (-A-) may be any chemical group or chemical moiety that can link -Y- to an amino acid unit. Alternatively, the second spacer unit may link -Y- to the first spacer unit if no amino acid unit is present. Alternatively, the second spacer unit may link -Y- to the cytotoxic moiety (-C) if neither the first spacer unit nor an amino acid unit is present. In this regard, -Y- is bound to the second spacer unit (-A-) as described herein. The second spacer unit (-A-) is bound to the amino acid unit (-W w-) and / or the presence or absence of a first spacer unit (-B-), the amino acid unit (-W w It may contain, or may be, a functional group capable of forming a bond to the amino acid unit (-B-), to the first spacer unit (-B-), or to the cytotoxic moiety (-CM). Preferably, the amino acid unit (-W w Functional groups that can form bonds to (-), particularly to the N-terminus of an amino acid unit, or to a first spacer unit (-B-), or to a cytotoxic moiety (-CM), for example, This is a carbonyl group, represented as TIFF2026513098000041.tif12128 or -C(O)-. The integer a associated with the second spacer unit may be 0 or 1. Preferably, the integer a is 1. Alternatively, in other embodiments, the second spacer unit does not exist (a=0).

[0186] Amino acid unit (-W) w -) If present, if the first spacer unit is present, the second spacer unit A may be linked to the first spacer unit B. Alternatively, if the first spacer unit is not present, the second spacer unit may be linked to the cytotoxic portion (CM) of the amino acid unit. Alternatively, if the second spacer unit is not present, Y may be linked to the first spacer unit of the amino acid unit. Alternatively, if neither the first nor the second spacer unit is present, Y may be linked to the cytotoxic portion of the amino acid unit.

[0187] Amino acid unit -W w - may be a dipeptide (w=2), tripeptide (w=3), tetrapeptide (w=4), pentapeptide (w=5), hexapeptide (w=6), heptapeptide (w=7), octapeptide (w=8), nonapeptide (w=9), decapeptide (w=10), undecapeptide (w=11), or dodecapeptide (w=12).

[0188] In some embodiments, the amino acid unit may include natural amino acids. In some embodiments, the amino acid unit may include non-natural amino acids.

[0189] In any of the embodiments described herein, each amino acid in an amino acid unit may exist independently in either an L-configuration or a D-configuration, with the exception of non-chiral amino acids, such as glycine. Preferably, in any of the embodiments described herein, each amino acid in an amino acid unit exists in an L-configuration (i.e., the native configuration), with the exception of non-chiral amino acids, such as glycine.

[0190] Preferably, if a second spacer unit (-A-) is present, in any one of the embodiments described herein, the amino acid unit -W w - The N-terminus is bonded to a second spacer unit (A), more preferably via the carbonyl group of the second spacer unit. Preferably, in any one of the embodiments described herein, the amino acid unit -W w - The C-terminus of - is bonded to the first spacer unit (B) if the first spacer unit is present, or in any one of the embodiments described herein, the amino acid unit -W w - The C-terminus may be bound to the cytotoxic moiety (-CM) if the first spacer unit is not present. In other embodiments, the amino acid unit -W w The N-terminus of - may be bonded to the first spacer unit (B) if present, and the C-terminus may be bonded to the second spacer unit A if present.

[0191] In some embodiments, w may be 1 or 2. Preferably, the amino acid unit W. w It is a dipeptide (w=2). In a dipeptide, each amino acid is independently represented by the formula shown in square brackets below: It may have TIFF2026513098000042.tif28128, in the formula, R 19Hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, The filename is TIFF2026513098000043.tif95145.

[0192] The amino acid unit can be enzymatically cleaved by one or more enzymes, non-limitingly including tumor-associated proteases, preferably cathepsins, more preferably cathepsin B, to release a cytotoxic moiety (-CM), which, in one embodiment, is protonated in vivo upon release to produce a free cytotoxic moiety (CM). Exemplary -W w -The units are expressed by formula (VII).

[0193] Therefore, -W w -The unit is given by formula (VII): It may be the dipeptide of TIFF2026513098000044.tif23128, where R 20 and R 21 The following: The filename is TIFF2026513098000045.tif64128.

[0194] An example of an amino acid unit is given by formula (VII) (wherein R 20 It is benzyl, and R 21 is -(CH2)4NH2(Phe-Lys); R 20 It is isopropyl, and R 21is -(CH2)4NH2(Val-Lys);R 20 It is isopropyl, and R 21 The units include, but are not limited to, those of -(CH2)3NHCONH2(Val-Cit).

[0195] Useful -W w -The units can be designed and optimized for selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases. In one embodiment, -W w -The units are catalyzed by cleavage by cathepsin B, cathepsin C, and / or cathepsin D, or plasmin proteases ("tumor-associated proteases"). Preferably, -W w- The unit is cleaved by cathepsin B. Suitable linkers that can be cleaved by proteases are, for example, GM Dubowchik et al., “Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconjugates; Model Studies of Enzymatic Drug Release and Antigen-Specific In Vitro Anticancer Activity”, Bioconjugate Chem., Vol. 13, No. 4, 2002, 855-869; SC Jeffrey et al., “Dipeptide-based highly potent doxorubicin antibody conjugate”, Bioorg. Med. Chem. Lett. 16 (2006), 358-362; and MS Kung Sutherland et al., “SGN-CD33A: a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML”, Blood, 22 August 2013, volume It is described in 122, number 8, 1455-1463.

[0196] R 19 , R 20 , or R 21 If R is anything other than hydrogen, 19 , R 20 , or R 21 The carbon atom to which it is bonded is chiral. 19 , R 20 , or R 21 Each carbon atom to which is bonded may exist independently in either an (S) or (R) configuration. Preferably, R 19 , R 20 , or R 21Each carbon atom to which the bond is located exists in a (S) configuration if it is chiral.

[0197] In one preferred embodiment, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC). In another preferred embodiment, the amino acid unit is valine-alanine (i.e., Val-Ala or VA). In yet another preferred embodiment, the amino acid unit is alanine-alanine (i.e., Ala-Ala or AA). In yet another preferred embodiment, the amino acid unit is phenylalanine-lysine (i.e., Phe-Lys or FK). Such connector units are exemplary examples of connector units that can be cleaved by proteases, such as cathepsin B.

[0198] Throughout this specification, the notation of peptides used herein follows conventional nomenclature. Thus, the N-terminus of a peptide is written on the left, and the C-terminus of the peptide is written on the right. As an exemplary but non-limiting example, in the dipeptide valine-citrulline (i.e., Val-Cit or VC), valine has the N-terminus and citrulline has the C-terminus. Preferably, in any one of the embodiments described herein, if a second spacer unit (-A-) is present, the N-terminus of the peptide, for example, the dipeptide (Val-Cit as an exemplary and non-limiting example), is bound to the second spacer unit (-A-), more preferably via the carbonyl group of the second spacer unit, and the C-terminus of the peptide is bound to the first spacer unit (-B-) if a first spacer unit (-B-) is present, or to the camptothecin moiety (-C) if a first spacer unit (-B-) is not present.

[0199] In yet another embodiment, the amino acid unit is N-methylvaline-citrulline. In yet another embodiment, the amino acid unit is selected from the group consisting of 5-aminovaleric acid, homophenylalanine-lysine, tetraisoquinoline carboxylate-lysine, cyclohexylalanine-lysine, isonepecotinic acid-lysine, β-alanine-lysine, and isonepecotinic acid.

[0200] Preferably, the amino acid unit is a dipeptide selected from the group consisting of valine-citrulline (i.e., Val-Cit or VC), valine-alanine (i.e., Val-Ala or VA), alanine-alanine (i.e., Ala-Ala or AA), and phenylalanine-lysine (i.e., Phe-Lys or FK). More preferably, the amino acid unit is a dipeptide selected from the group consisting of valine-citrulline (i.e., Val-Cit or VC), valine-alanine (i.e., Val-Ala or VA), and phenylalanine-lysine (i.e., Phe-Lys or FK). Even more preferably, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC) or valine-alanine (i.e., Val-Ala or VA). Even more preferably, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC).

[0201] In some embodiments, the amino acid unit is selected from the group consisting of valine-glutamine (i.e., Val-Gln or VQ), leucine-glutamine (i.e., Leu-Gln or LQ), phenylalanine-glutamine (i.e., Phe-Gln or FQ), and threonine-threonine (i.e., Thr-Thr or TT). Preferably, the amino acid unit is selected from the group consisting of valine-glutamine (i.e., Val-Gln or VQ), leucine-glutamine (i.e., Leu-Gln or LQ), and phenylalanine-glutamine (i.e., Phe-Gln or FQ). More preferably, the amino acid unit is valine-glutamine (i.e., Val-Gln or VQ) or leucine-glutamine (i.e., Leu-Gln or LQ). Connector units comprising amino acid units according to these embodiments may be exemplary examples of connector units that can be cleaved in particular by proteases, such as cathepsins (e.g., cathepsin B). Amino acid units according to these embodiments and further suitable amino acid units are disclosed, for example, in Salomon et al., “Optimizing Lysosomal Activation of Antibody-Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers”, Mol. Pharmaceutics 2019, 16, 12, 4817-4825.

[0202] The first spacer unit (B) is, if present, an amino acid unit (W) if present. w The cytotoxic portion may be linked to the first spacer unit (B). Alternatively, if no amino acid unit is present, the second spacer unit (A) may be linked to the cytotoxic portion (CM). If neither an amino acid unit nor the second spacer unit is present, the first spacer unit may be linked to the cytotoxic portion (Y).

[0203] The integer b may be 0 or 1. In a preferred embodiment, the integer b is 1. Alternatively, in another embodiment, the integer b is 0, and the first spacer unit does not exist.

[0204] The first spacer unit (-B-) may be of two common types: self-destructive and non-self-destructive. The non-self-destructive first spacer unit is the amino acid unit (-W) of the linker (L). w After cleavage of -), particularly enzymatic cleavage, a portion or all of the first spacer unit remains bound to the cytotoxic moiety (CM). Alternatively, exemplary compounds containing a self-destructive first spacer unit can release the cytotoxic moiety -CM without requiring a separate hydrolysis step. In exemplary embodiments, the self-destructive first spacer unit is bound to the -W via the amino nitrogen atom of the PAB group. w This is a PAB group that is linked to - and directly connected to -CM via a carbonate, carbamate, or ether group. While not bound by any particular theory or mechanism, Scheme 2 illustrates a possible mechanism of drug release by a PAB group directly linked to the drug moiety -D via a carbamate or carbonate group, as adopted by Toki et al. (2002) J Org. Chem. 67:1866-1872. In this specification, the drug moiety D is also referred to as the cytotoxic moiety CM. In formula TIFF2026513098000046.tif102128, Q is a -(C1~C8)alkyl, -O-(C1~C8)alkyl, -halogen, -nitro, or -cyano; m is an integer in the range of 0 to 4, preferably m is 0, 1, or 2, more preferably m is 0 or 1, and even more preferably m is 0; and p is in the range of 1 to 20.

[0205] While not adhering to any particular theory or mechanism, Scheme 3 illustrates a possible mechanism of drug release by a PAB group directly bound to drug moiety-D via an ether or amine bond. In this specification, drug moiety-D is also referred to as cytotoxic moiety-CM. In formula TIFF2026513098000047.tif111128, Q is a -(C1~C8)alkyl, -O-(C1~C8)alkyl, -halogen, -nitro, or -cyano; m is an integer in the range of 0 to 4, preferably m is 0, 1, or 2, more preferably m is 0 or 1, and even more preferably m is 0; and p is in the range of 1 to 20.

[0206] Other examples of self-destructing spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho-aminobenzyl acetals or para-aminobenzyl acetals. Spacers that undergo cyclization upon hydrolysis of the amide bond, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J. Amer. Chem. Soc., 1972, 94, 5815), and 2-aminophenylpropionic acid amide (Amsberry, et al., J. Org. Chem., 1990, 55, 5867), may be used. The elimination of amine-containing drugs substituted at the α-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) is also an example of a useful self-destructing spacer in the exemplary compounds.

[0207] In one embodiment, the first spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit shown in Scheme 4, which can be used to incorporate and release multiple drug(D) units. In this specification, drug portion D is also referred to as cytotoxic portion CM. In formula TIFF2026513098000048.tif44128, Q is a -(C1~C8)alkyl, -O-(C1~C8)alkyl, -halogen, -nitro, or -cyano; m is an integer in the range of 0 to 4, preferably m is 0, 1, or 2; more preferably m is 0 or 1; even more preferably m is 0; p is in the range of 1 to 10; n is 0 or 1; and p is in the range of 1 to 20.

[0208] In a preferred embodiment, the first spacer unit is given by equation (X): Represented by TIFF2026513098000049.tif27128, In the formula, Q is a -(C1~C8)alkyl, -O-(C1~C8)alkyl, -halogen, -nitro, or -cyano; m is an integer in the range of 0 to 4, preferably m is 0, 1, or 2; more preferably m is 0 or 1; and in a very preferred embodiment, m is 0. Preferably, in formula (X), where an amino acid unit is present, the NH group is attached to the C-terminus of the amino acid unit. Preferably, in formula (X), the C(O) group is attached to a cytotoxic moiety (CM), such as a camptothecin moiety.

[0209] In a very preferred embodiment, the first spacer unit has the following structure: It is a PAB group having TIFF2026513098000050.tif29128. Preferably, if an amino acid unit is present, the NH group has an amino acid unit (-W w -) is more preferably attached to the C-terminus of an amino acid unit. Preferably, the C(O) group is attached to the cytotoxic moiety (CM), for example, the camptothecin moiety.

[0210] In some embodiments, the first spacer group (-B-) is a “self-destructing moiety” of a heterocyclic system of formula I, II, or III, conjugated to the cytotoxic moiety, and contains an amide group. This amide group initiates a reaction when hydrolyzed by an intracellular protease, which ultimately cleaves the first spacer unit (-B-) from the cytotoxic moiety, resulting in the release of the cytotoxic moiety from the conjugate in an active form. The connector unit is an amino acid unit (-W) adjacent to the first spacer group (-B-). w -) further contains the amino acid unit (-W w -) is a substrate for intracellular enzymes that cleave the peptide at an amide bond shared with the first spacer group (-B-), such as intracellular proteases, such as cathepsins (e.g., cathepsin B). The self-destructing portion of the heterocyclic system is described, for example, in WO 2019 / 236954.

[0211] In some embodiments, the first spacer unit (-B-) is given by formulas I, II, and III: A self-destructing group of a heterocyclic system selected from TIFF2026513098000051.tif77128, In the formula, the dashed line represents the amino acid unit -W. w - Indicates a covalent binding site to the cytotoxic moiety CM, where U is O, S, or NR. 6 Q is CR 4 or N; V 1 , V 2 , and V 3 CR independently 4 or N, except in the case of equations II and III, Q, V 1 , and V 2 At least one of them is N; T may be O hanging from the cytotoxic part (-CM); R 1 , R 2 , R 3 , and R 4 H, F, Cl, Br, I, OH, -N(R 5 )2, -N(R 5 )3 +-(C1~C8) alkyl halide, carboxylate, sulfate, sulfamate, sulfonate, -SO2R 5 -S(=O)R 5 , -SR 5 , -SO2N(R 5 )2, -C(=O)R 5 , -CO2R 5 -C(=O)N(R 5 )2, -CN, -N3, -NO2, -(C1~C8)alkoxy, -(C1~C8)halosubstituted alkyl, polyethylene oxy, phosphonate, phosphate, -(C1~C8)alkyl, -(C1~C8)substituted alkyl, -(C2~C8)alkenyl, -(C2~C8)substituted alkenyl, -(C2~C8)alkynyl, -(C2~C8)substituted alkynyl, -(C6~C 20 )aryl, -(C6~C 20 ) Substitute aryl, -(C3~C 20 )Hybrid algebras, and -(C3~C 20 ) If independently selected from a group consisting of substitutional heteroalgs; or if they combine, R 2 and R 3 It forms a carbonyl (=O) or a spirocyclic carbon containing 3 to 7 carbon atoms; R 5 and R 6 H, -(C1~C8)alkyl, -(C1~C8)substituted alkyl, -(C2~C8)alkenyl, -(C2~C8)substituted alkenyl, -(C2~C8)alkynyl, -(C2~C8)substituted alkynyl, -(C6~C 20 )aryl, -(C6~C 20 ) Substitute aryl, -(C3~C 20 )Hybrid algebras, and -(C3~C 20 ) Independently selected from substituted heterocycles; where -(C1~C8) substituted alkyl, -(C2~C8) substituted alkenyl, -(C2~C8) substituted alkynyl, -(C6~C 20 ) Substitute aryl, and -(C3~C 20 ) Substitutive heterocycles include F, Cl, Br, I, OH, -N(R 5 )2, -N(R 5 )3 +-(C1~C8) alkyl halide, carboxylate, sulfate, sulfamate, sulfonate, -(C1~C8) alkyl sulfonate, -(C1~C8) alkylamino, 4-dialkylaminopyridinium, -(C1~C8) alkylhydroxy, -(C1~C8) alkylthiol, -SO2R 5 -S(=O)R 5 , -SR 5 , -SO2N(R 5 )2, -C(=O)R 5 , -CO2R 5 -C(=O)N(R 5 )2, -CN, -N3, -NO2, -(C1~C8)alkoxy, -(C1~C8)trifluoroalkyl, -(C1~C8)alkyl, -(C3~C 12 ) carbocycle, -(C6~C 20 )Aaryl, -(C3~C 20 ) It is independently substituted with one or more substituents selected from the group consisting of heterocycles, polyethylene oxy, phosphonates, and phosphates.

[0212] Antibody-drug conjugates (ADCs) containing a heterocyclic, self-destructing moiety are stable extracellularly or in the absence of enzymes capable of cleaving the amide bond of the self-destructing moiety. However, upon entering a cell or exposure to a suitable enzyme, the amide bond is cleaved, initiating a spontaneous self-destruction reaction. This results in the cleavage of the bond covalently linking the self-destructing moiety to the camptothecin moiety, thereby releasing the drug in an underivativeed or pharmacologically active form.

[0213] The self-destructing portion in the conjugate may further contain one or more heteroatoms, thereby potentially providing improved solubility, potentially improving cleavage rate, and / or reducing the aggregation tendency of the antibody-drug conjugate (ADC). Thus, self-destructing connector unit constructs of heterocyclic systems may, in some cases, result in increased efficacy, decreased toxicity, and / or desirable pharmacokinetic and / or pharmacodynamic properties.

[0214] When the cytotoxic moiety CM is the camptothecin moiety, T in formulas I-III is understood to be, for example, O. This is because T may originate from the tertiary hydroxyl (-OH) present in the lactone ring portion of the camptothecin moiety. When the cytotoxic moiety CM is the camptothecin moiety, T in formulas I-III may also be, for example, NH. This is because T may originate from the amino group (-NH2) of the camptothecin moiety, for example, exatecan.

[0215] While not limited to any specific theory or mechanism, the presence of electron-withdrawing groups in the heterocycle of equations I, II, or III can sometimes slow down the cleavage rate.

[0216] In one embodiment, the self-destructing portion is a group of formula I, where Q is N and U is O or S. Such a group has a nonlinear structural feature that improves the solubility of the conjugate. In this context, R is sometimes H, methyl, nitro, or CF3. In one embodiment, Q is N and U is O, thereby forming an oxazole ring and R is H. In another embodiment, Q is N and U is S, thereby forming a thiazole ring, which may be substituted with a Me group or a CF3 group at the R position.

[0217] In another illustrative embodiment, the self-destructing part is such that Q is N and V 1 and V 2 The base of formula II is independently N or CH. In another embodiment, Q, V 1 , and V 2 These are N, respectively. In another embodiment, Q and V 1 is N, V 2 is CH. In another embodiment, Q and V 2 is N, V 1 is CH. In another embodiment, Q and V 1 Both are CH, V 2 In another embodiment, Q is N and V1 and V 2 Both are CH.

[0218] In another aspect, the self-destructing parts are Q, V 1 , V 2 , and V 3 However, each is independently N or CH, which are the bases of equation III. In another embodiment, Q is N and V 1 , V 2 , and V 3 These are N, respectively. In another embodiment, Q, V 1 , and V 2 These are CH and V 3 is N. In another embodiment, Q, V 2 , and V 3 These are CH and V 1 is N. In another embodiment, Q, V 1 , and V 3 These are CH and V 2 is N. In another embodiment, Q and V 2 Both are N, V 1 and V 3 Both are CH. In another embodiment, Q and V 2 Both are CH, V 1 and V 3 Both are N. In another aspect, Q and V 3 Both are N, V 1 and V 2 Both are CH.

[0219] Preferably, the connector unit (CU) is given by the formula: * -A a -W w -B b - ## The formula has such that the integer a is 1, the integer b is 1, and the integer w is 2, 3, or 4, more preferably the integer w is 2 or 3; in a very preferred embodiment, the integer w is 2; -A-, each -W-, and -B- are as defined herein; *indicates a binding site to Y; ## indicates a binding site to the cytotoxic moiety (CM).

[0220] Preferably, the connector unit (CU) has the following structure: * -A a -W w -B b - ## It has, In the formula, -A- is a second spacer unit as described herein; a is an integer as described herein; preferably a is 1; -B- is a first spacer unit as described herein; b is an integer as described herein; preferably b is 1; * indicates a binding site to Y; ## indicates a binding site to the cytotoxic region (-CM); -W w - is a dipeptide selected from the group consisting of valine-citrulline (i.e., Val-Cit or VC), valine-alanine (i.e., Val-Ala or VA), alanine-alanine (i.e., Ala-Ala or AA), and phenylalanine-lysine (i.e., Phe-Lys or FK). Preferably, in these embodiments, the amino acid unit is a dipeptide selected from the group consisting of valine-citrulline (i.e., Val-Cit or VC), valine-alanine (i.e., Val-Ala or VA), and phenylalanine-lysine (i.e., Phe-Lys or FK). More preferably, in these embodiments, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC) or valine-alanine (i.e., Val-Ala or VA). Even more preferably, in these embodiments, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC). Or, in these embodiments, the amino acid unit -W w- may be a dipeptide selected from the group consisting of valine-glutamine (i.e., Val-Gln or VQ), leucine-glutamine (i.e., Leu-Gln or LQ), phenylalanine-glutamine (i.e., Phe-Gln or FQ), and threonine-threonine (i.e., Thr-Thr or TT). In these embodiments, the amino acid unit may be a dipeptide selected from the group consisting of valine-glutamine (i.e., Val-Gln or VQ), leucine-glutamine (i.e., Leu-Gln or LQ), and phenylalanine-glutamine (i.e., Phe-Gln or FQ). In these embodiments, the amino acid unit may be valine-glutamine (i.e., Val-Gln or VQ) or leucine-glutamine (i.e., Leu-Gln or LQ). Connector units (CUs) according to these embodiments may be exemplary examples of connector units that can be cleaved in particular by proteases, such as cathepsins (e.g., cathepsin B).

[0221] Preferably, the connector unit CU has the following structure: It has TIFF2026513098000052.tif31128, In the formula, -A- is a second spacer unit as described herein; a is an integer as described herein; preferably a is 1; -W w - is an amino acid unit as described herein; w is an integer as described herein; preferably w is 2, 3, or 4 (i.e., preferably -W) w - is a dipeptide, tripeptide, or tetrapeptide), more preferably w is 2 or 3 (i.e., more preferably -W). w - is a dipeptide or tripeptide), for example, w may be 1 or 2; in a very preferred embodiment, w is 2 (i.e., even more preferably -W) w - is a dipeptide); Q is as defined herein; m is an integer as defined herein, preferably m is 0; * indicates a binding site to Y; ## indicates a binding site to the cytotoxic moiety (-CM). Preferably, in these embodiments, the amino acid unit -W w - is a dipeptide selected from the group consisting of valine-citrulline (i.e., Val-Cit or VC), valine-alanine (i.e., Val-Ala or VA), alanine-alanine (i.e., Ala-Ala or AA), and phenylalanine-lysine (i.e., Phe-Lys or FK). More preferably, in these embodiments, the amino acid unit is a dipeptide selected from the group consisting of valine-citrulline (i.e., Val-Cit or VC), valine-alanine (i.e., Val-Ala or VA), and phenylalanine-lysine (i.e., Phe-Lys or FK). Even more preferably, in these embodiments, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC) or valine-alanine (i.e., Val-Ala or VA). Even more preferably, in these embodiments, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC). Or, in these embodiments, the amino acid unit -W w- may be a dipeptide selected from the group consisting of valine-glutamine (i.e., Val-Gln or VQ), leucine-glutamine (i.e., Leu-Gln or LQ), phenylalanine-glutamine (i.e., Phe-Gln or FQ), and threonine-threonine (i.e., Thr-Thr or TT). In these embodiments, the amino acid unit may be a dipeptide selected from the group consisting of valine-glutamine (i.e., Val-Gln or VQ), leucine-glutamine (i.e., Leu-Gln or LQ), and phenylalanine-glutamine (i.e., Phe-Gln or FQ). In these embodiments, the amino acid unit may be valine-glutamine (i.e., Val-Gln or VQ) or leucine-glutamine (i.e., Leu-Gln or LQ). Connector units (CUs) according to these embodiments may be exemplary examples of connector units (CUs) that can be cleaved in particular by a protease, such as a cathepsin (e.g., cathepsin B).

[0222] More preferably, the connector unit CU has the following structure: It has TIFF2026513098000053.tif36128, During the ceremony, TIFF2026513098000054.tif29128 is as defined herein; * indicates a bond point to Y; # indicates the amino acid unit -W if present. w - Indicates a bonding site to or to an NH group; -W w - is an amino acid unit as described herein; w is an integer as described herein, preferably w is 2, 3, or 4 (i.e., preferably -W). w - is a dipeptide, tripeptide, or tetrapeptide), more preferably w is 2 or 3 (i.e., more preferably -W). w - is a dipeptide or tripeptide), and in a very preferred embodiment, w is 2 (i.e., even more preferably -W).w - is a dipeptide); Q is as defined herein; m is an integer as defined herein, preferably m is 0; * indicates a binding site to Y; ## indicates a binding site to the cytotoxic moiety (-CM). Preferably, in these embodiments, the amino acid unit -W w - is a dipeptide selected from the group consisting of valine-citrulline (i.e., Val-Cit or VC), valine-alanine (i.e., Val-Ala or VA), alanine-alanine (i.e., Ala-Ala or AA), and phenylalanine-lysine (i.e., Phe-Lys or FK). More preferably, in these embodiments, the amino acid unit is a dipeptide selected from the group consisting of valine-citrulline (i.e., Val-Cit or VC), valine-alanine (i.e., Val-Ala or VA), and phenylalanine-lysine (i.e., Phe-Lys or FK). Even more preferably, in these embodiments, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC) or valine-alanine (i.e., Val-Ala or VA). Even more preferably, in these embodiments, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC). Or, in these embodiments, the amino acid unit -W w- may be a dipeptide selected from the group consisting of valine-glutamine (i.e., Val-Gln or VQ), leucine-glutamine (i.e., Leu-Gln or LQ), phenylalanine-glutamine (i.e., Phe-Gln or FQ), and threonine-threonine (i.e., Thr-Thr or TT). In these embodiments, the amino acid unit may be a dipeptide selected from the group consisting of valine-glutamine (i.e., Val-Gln or VQ), leucine-glutamine (i.e., Leu-Gln or LQ), and phenylalanine-glutamine (i.e., Phe-Gln or FQ). In these embodiments, the amino acid unit may be valine-glutamine (i.e., Val-Gln or VQ) or leucine-glutamine (i.e., Leu-Gln or LQ). Connector units (CUs) according to these embodiments may be exemplary examples of connector units that can be cleaved in particular by proteases, such as cathepsins (e.g., cathepsin B).

[0223] More preferably, the connector unit CU has the following structure: It has TIFF2026513098000055.tif35128, During the ceremony, -W w - is an amino acid unit as described herein; w is an integer as described herein, preferably w is 2, 3, or 4 (i.e., preferably -W). w - is a dipeptide, tripeptide, or tetrapeptide), more preferably w is 2 or 3 (i.e., more preferably -W). w - is a dipeptide or tripeptide), and in a very preferred embodiment, w is 2 (i.e., even more preferably -W). w - is a dipeptide); * indicates a binding site to Y; ## indicates a binding site to the cytotoxic moiety (-CM). Preferably, in these embodiments, the amino acid unit -W w- is a dipeptide selected from the group consisting of valine-citrulline (i.e., Val-Cit or VC), valine-alanine (i.e., Val-Ala or VA), alanine-alanine (i.e., Ala-Ala or AA), and phenylalanine-lysine (i.e., Phe-Lys or FK). More preferably, in these embodiments, the amino acid unit is a dipeptide selected from the group consisting of valine-citrulline (i.e., Val-Cit or VC), valine-alanine (i.e., Val-Ala or VA), and phenylalanine-lysine (i.e., Phe-Lys or FK). Even more preferably, in these embodiments, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC) or valine-alanine (i.e., Val-Ala or VA). Even more preferably, in these embodiments, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC). Or, in these embodiments, the amino acid unit -W w - may be a dipeptide selected from the group consisting of valine-glutamine (i.e., Val-Gln or VQ), leucine-glutamine (i.e., Leu-Gln or LQ), phenylalanine-glutamine (i.e., Phe-Gln or FQ), and threonine-threonine (i.e., Thr-Thr or TT). In these embodiments, the amino acid unit may be a dipeptide selected from the group consisting of valine-glutamine (i.e., Val-Gln or VQ), leucine-glutamine (i.e., Leu-Gln or LQ), and phenylalanine-glutamine (i.e., Phe-Gln or FQ). In these embodiments, the amino acid unit may be valine-glutamine (i.e., Val-Gln or VQ) or leucine-glutamine (i.e., Leu-Gln or LQ). Connector units (CUs) according to these embodiments may be exemplary examples of connector units that can be cleaved in particular by proteases, such as cathepsins (e.g., cathepsin B).

[0224] In a preferred embodiment, the connector unit CU is the amino acid unit -W w - Containing the dipeptide valine-citrulline, the following structure: TIFF2026513098000056.tif52130 is included, in the formula, * indicates a binding point to Y; ## indicates a binding point to the cytotoxic moiety (-CM). Such connector units are exemplary examples of connector units that can be cleaved, in particular, by proteases, such as cathepsins (e.g., cathepsin B).

[0225] In another preferred embodiment, the connector unit CU is the amino acid unit -W w - Containing the dipeptide valine-alanine, the following structure: TIFF2026513098000057.tif38130 is included, in the formula, * indicates a binding site to Y; ## indicates a binding site to the cytotoxic moiety (-CM). Such connector units (CUs) are exemplary examples of connector units that can be cleaved, in particular, by proteases, such as cathepsins (e.g., cathepsin B).

[0226] The connector unit CU has the following structure: It may have TIFF2026513098000058.tif27128, During the ceremony, TIFF2026513098000059.tif26128 is as defined herein; * indicates the bond point to Y; # indicates the amino acid unit -W w - indicates the connection point to; -W w - is an amino acid unit as described herein; w is an integer as described herein, preferably w is 2, 3, or 4 (i.e., preferably -W). w - is a dipeptide, tripeptide, or tetrapeptide), more preferably an integer w is 2 or 3 (i.e., more preferably -W w- is a dipeptide or tripeptide), and more preferably w is 2 (i.e., more preferably -W). w - is a dipeptide); * indicates a binding site to Y; ## indicates a binding site to the cytotoxic moiety (-CM). Preferably, in these embodiments, the amino acid unit -W w - is a dipeptide selected from the group consisting of valine-citrulline (i.e., Val-Cit or VC), valine-alanine (i.e., Val-Ala or VA), alanine-alanine (i.e., Ala-Ala or AA), and phenylalanine-lysine (i.e., Phe-Lys or FK). More preferably, in these embodiments, the amino acid unit is a dipeptide selected from the group consisting of valine-citrulline (i.e., Val-Cit or VC), valine-alanine (i.e., Val-Ala or VA), and phenylalanine-lysine (i.e., Phe-Lys or FK). Even more preferably, in these embodiments, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC) or valine-alanine (i.e., Val-Ala or VA). Even more preferably, in these embodiments, the amino acid unit is valine-citrulline (i.e., Val-Cit or VC). Or, in these embodiments, the amino acid unit -W w- may be a dipeptide selected from the group consisting of valine-glutamine (i.e., Val-Gln or VQ), leucine-glutamine (i.e., Leu-Gln or LQ), phenylalanine-glutamine (i.e., Phe-Gln or FQ), and threonine-threonine (i.e., Thr-Thr or TT). In these embodiments, the amino acid unit may be a dipeptide selected from the group consisting of valine-glutamine (i.e., Val-Gln or VQ), leucine-glutamine (i.e., Leu-Gln or LQ), and phenylalanine-glutamine (i.e., Phe-Gln or FQ). In these embodiments, the amino acid unit may be valine-glutamine (i.e., Val-Gln or VQ) or leucine-glutamine (i.e., Leu-Gln or LQ).

[0227] In some embodiments, the connector unit CU is the amino acid unit -W w - Containing the dipeptide valine-citrulline, the following structure: It may have TIFF2026513098000060.tif39128, in the formula, * indicates a binding site to Y; ## indicates a binding site to the cytotoxic region (-CM).

[0228] In some embodiments, the connector unit CU is the amino acid unit -W w - Containing the dipeptide valine-alanine, the following structure: It may have TIFF2026513098000061.tif28128, in the formula, * indicates a binding site to Y; ## indicates a binding site to the cytotoxic region (-CM).

[0229] The connector unit (-CU-) has the following structure: It may have TIFF2026513098000062.tif22128, in the formula, TIFF2026513098000063.tif22128 is as defined herein; *indicates a binding site to Y; # indicates a binding site to the cytotoxic region (-CM).

[0230] In some embodiments, the connector unit CU has the following structure: It may have TIFF2026513098000064.tif25128, in the formula, * indicates a binding site to Y; # indicates a binding site to the cytotoxic region (-CM).

[0231] Cytotoxic moiety (-CM) This disclosure provides antibody-drug conjugates (ADCs) comprising a cytotoxic moiety. The terms “cytotoxic moiety,” “drug,” “drug moiety,” “payload,” or “cytotoxic payload” may be used synonymously and, as used herein, mean the chemical or biochemical moiety conjugated to an anti-TPBG antibody (Ab) or its antigen-binding fragment. In this regard, the antibody-drug conjugate (ADC) of formula (I) described herein is again referred to. The antibody (Ab) can be conjugated with several identical or different cytotoxic moieties using any method described herein or known in the art. In some embodiments, the cytotoxic moiety may be a molecule that has cytotoxic effects on mammalian cells, can induce apoptosis, and / or can have a regulatory effect on malignant cells. The cytotoxic moiety may be hydrophobic.

[0232] In some preferred embodiments, the cytotoxic moiety is an anticancer agent. Therefore, the cytotoxic moiety may be selected from the group consisting of camptothecin, mytansinoids, calicheamycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof.

[0233] Camptothecin portion Preferably, the cytotoxic moiety CM is the camptothecin moiety. The term “camptothecin moiety” includes camptothecin itself and its analogues. Camptothecin is a topoisomerase inhibitor and was discovered in 1966 by ME Wall and MC Wani in a systematic screening of natural products for anticancer drugs. Camptothecin was isolated from the bark and trunk of Camptotheca acuminata (Camptotheca genus, also known as the "Joy Tree"), a tree native to China used as a cancer treatment in traditional Chinese medicine. Camptothecin has the following structure: It has TIFF2026513098000065.tif25128.

[0234] The term "camptothecin moiety" also includes analogues of camptothecin. In this regard, the term "camptothecin moiety" refers to the structure of camptothecin: This means any portion that includes TIFF2026513098000066.tif25128 and may be optionally substituted. Any substituents include, as an illustrative and non-limiting example, (C1~C 10The camptothecin moiety may include alkyl, (C3-C8) carbocyclo, (C3-C8) heterocyclo, aryl, amino, hydroxyl, carbonyl, amide, ester, carbamate, carbonate, and / or silyl groups. The camptothecin moiety may have one or more functional groups capable of forming a bond with linker L. Those skilled in the art will readily select a suitable camptothecin moiety having the desired biological activity. Camptothecin analogs, such as topotecan, irinotecan, or berotecan, are approved and currently used in cancer chemotherapy.

[0235] The following camptothecin analogs are also referred to by the term "camptothecin moiety." Further camptothecin analogs that can be used as the camptothecin moiety are described in WO 2019 / 236954 and EP 0 495 432.

[0236] In some embodiments, the camptothecin moiety (CM) is selected from the group consisting of exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, berotecan, lulutotecan, rubitecan, siratecan, cositecan, and gimatecan. Preferably, the camptothecin moiety is selected from the group consisting of exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, and berotecan. SN38 has the following structure: The structures of exatecan, camptothecin, topotecan, irinotecan, and belotecan, which have TIFF2026513098000069.tif22128, are as described herein.

[0237] More preferably, in any of the embodiments described herein, the camptothecin portion CM has the following structure: This is an exatecan containing TIFF2026513098000070.tif55128.

[0238] More preferably, the camptothecin moiety (CM) has the following structure: This is an exatecan containing TIFF2026513098000071.tif55128.

[0239] Preferably, in any one of these embodiments, the exatecan is bonded to the connector unit CU via an amino group (i.e., via the NH2 group of the exatecan). When the exatecan is bonded to the connector unit CU via an amino group, the hydrogen atoms of the amino group of the exatecan are substituted by the connector unit CU. Therefore, the exatecan bonded to the connector unit CU via an amino group is, for example, as follows: It can be expressed as TIFF2026513098000072.tif59166, where # indicates the connection point to the connector unit CU.

[0240] In some aspects / modes, the present invention also relates to formula (I): The same applies to TIFF2026513098000073.tif34128 or conjugates having pharmaceutically acceptable salts or solvates thereof. During the ceremony, Ab is the anti-TPBG antibody described herein; TIFF2026513098000074.tif6128 is either a double bond or TIFF2026513098000075.tif6128 is a single bond; If TIFF2026513098000076.tif6128 has a double bond, then V does not exist, or If TIFF2026513098000077.tif5128 is a single bond, then V is H; If TIFF2026513098000078.tif6128 is a double bond, then X is R3-C, or If TIFF2026513098000079.tif6128 is a single join, then X is: The filename is TIFF2026513098000080.tif9128; Y is NH; R 1 Structure: This is a polyethylene glycol unit having TIFF2026513098000081.tif15128. During the ceremony, TIFF2026513098000082.tif9128 indicates the position of O, K F This is as defined herein, preferably K F is H and o is an integer as defined herein, preferably an integer in the range of 8 to 30, more preferably 16 to 30, even more preferably 20 to 28, even more preferably o is 22, 23, 24, 25, or 26, even more preferably o is 23, 24, or 25, and even more preferably o is 24; R 3 is H; R 4 is H; The CU has the following structure: This is a connector unit having TIFF2026513098000083.tif50128, where # indicates the connection point to Y, * This indicates the binding site to the camptothecin moiety (CM); CM is the camptothecin part; m is 1; and n is an integer as defined herein; Preferably, n is an integer in the range of 1 to 10, more preferably 2 to 10, even more preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, and even more preferably n is 8 or Preferably, n is an integer in the range of 1 to 10, more preferably 2 to 8, even more preferably 3 to 6, even more preferably n is 4 or 5, and even more preferably n is 4.

[0241] Preferably, TIFF2026513098000084.tif6128 has a double bond; V is absent; X is R3-C; and R 3 H is H.

[0242] In some embodiments, TIFF2026513098000085.tif6128 may be a single bond; V may be H; X is TIFF2026513098000086.tif9128 is fine; R 3 is H and R 4 It can be H.

[0243] Preferably, the camptothecin portion CM has the following structure: This is an exatecan containing TIFF2026513098000087.tif55128.

[0244] More preferably, the camptothecin moiety has the following structure: This is an exatecan containing TIFF2026513098000088.tif55128.

[0245] Preferably, in any one of these embodiments, the exatecan is bonded to the connector unit CU via an amino group.

[0246] In some aspects / modes, the present invention also uses the following formula (Ia): Regarding antibody-drug conjugates (ADCs) containing TIFF2026513098000089.tif74151, During the ceremony, Ab is the anti-TPBG antibody described herein; and n is an integer a as defined herein: Preferably, n is an integer in the range of 1 to 10, more preferably 2 to 10, even more preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, and even more preferably n is 8 or Preferably, n is an integer in the range of 1 to 10, more preferably 2 to 8, even more preferably 3 to 6, even more preferably n is 4 or 5, and even more preferably n is 4.

[0247] Auristatin In some embodiments, the cytotoxic moiety (CM) is auristatin. Preferably, the auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE). More preferably, the auristatin is monomethyl auristatin E (MMAE).

[0248] In some embodiments, the cytotoxic moiety CM is monomethyl auristatin F (also known as MMAF). MMAF has the following structural formula: Represented by TIFF2026513098000090.tif34128.

[0249] Preferably, MMAF is an asterisk (" * It is bonded to the connector unit CU via the N-terminus indicated by ''. Therefore, if MMAF is bonded to the connector unit CU via the N-terminus, the hydrogen atom at the N-terminus of MMAF is substituted by the connector unit CU.

[0250] In some embodiments, the auristatin drug portion is monomethyl auristatin E (also known as MMAE). MMAE has the following structural formula: Represented by TIFF2026513098000091.tif38128.

[0251] Preferably, MMAE is an asterisk (" * It is bonded to the connector unit CU via the N-terminus indicated by ''. Therefore, when MMAE is bonded to the connector unit CU via the N-terminus, the hydrogen atom at the N-terminus of MMAE is substituted by the connector unit CU.

[0252] These molecules have been shown to non-competitively inhibit the binding of vincristine to tubulin (at a location known as the vinca / peptide domain), but to the RZX / MAY domain.

[0253] Compound of formula (II) In some aspects / applications, the present invention also relates to formula (II): The same applies to compounds having TIFF2026513098000092.tif29128 or pharmaceutically acceptable salts or solvates thereof. During the ceremony, TIFF2026513098000093.tif6128 is either a triple bond or TIFF2026513098000094.tif6128 is a double bond; If TIFF2026513098000095.tif6128 has a triple bond, then V does not exist, or If TIFF2026513098000096.tif6128 has a double bond, then V is either H or (C1-C8) alkyl; If TIFF2026513098000097.tif6128 has a triple bond, then X is R3-C, or If TIFF2026513098000098.tif6128 has a double bond, then X is: The filename is TIFF2026513098000099.tif9128; Y is NR 5 , S, O, or CR 6 R 7 and; R 1 is an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 3 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 4 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 5is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 6 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 7 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; CU stands for connector unit; CM is the cytotoxic part; and m is an integer in the range of 1 to 10.

[0254] Preferably, R 3 is H or (C1-C8) alkyl, more preferably R 3 is H. Preferably, R 4 If present, is H or (C1-C8) alkyl; more preferably R 4 If present, it is H. Preferably, R 5 If present, is H or (C1-C8) alkyl; more preferably R 5 If present, it is H. Preferably, R 6 If present, is H or (C1-C8) alkyl; more preferably R 6 If present, it is H. Preferably, R 7 If present, is H or (C1-C8) alkyl; more preferably R 7 If it exists, it is H.

[0255] Preferably, TIFF2026513098000100.tif6128 has a triple bond; V is absent; X is R3-C; and R 3 is H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 3 is H or (C1-C8) alkyl; more preferably, R 3 H is H.

[0256] more, TIFF2026513098000101.tif6128 represents a triple bond; V is absent; X represents R3-C, and R3 represents H or (C1-C8) alkyl. Preferably, R3 represents H or (C1-C6) alkyl, more preferably H or (C1-C4) alkyl, and even more preferably H or (C1-C2) alkyl. Even more preferably, R3 is H.

[0257] In some embodiments, TIFF2026513098000102.tif6128 may be a double bond; V is H or (C1~C8) alkyl, preferably V is H; X is TIFF2026513098000103.tif9128; R3 is H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; more preferably, R 3 is H or (C1-C8) alkyl, more preferably R 3 H is R 4 is H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 4 is H or (C1-C8) alkyl, preferably R 4 H is H.

[0258] In some embodiments, TIFF2026513098000104.tif6128 may represent a double bond; V may be H or (C1~C8) alkyl; X is R3 may represent TIFF2026513098000105.tif9128; and R3 and R4 may independently represent H or (C1-C8) alkyl. Preferably, R3 and R4 independently represent H or (C1-C6) alkyl, more preferably H or (C1-C4) alkyl, and even more preferably H or (C1-C2) alkyl. Preferably, R3 and R4 are the same; and even more preferably, R3, R4, and V are the same. Even more preferably, R3 and R4 are both H. Preferably, V is H or (C1-C6) alkyl, more preferably H or (C1-C4) alkyl, and even more preferably H or (C1-C2) alkyl. Even more preferably, V is H. In a preferred embodiment, R3, R4, and V are each H.

[0259] In any one of the compounds of formula (II), any variable may be defined as described herein, specifically, as in the case of the antibody-drug conjugate (ADC) of formula (I) and / or the molecule containing the thiol of formula (III). Thus, Ab, TIFF2026513098000106.tif6128, V, X, Y, R 1 , R 3 , R 4 , R 5 , R 6 , R 7 CU, CM, m, and n may be as defined herein. Preferably, Y is NH.

[0260] Method for preparing antibody-drug conjugates (ADCs) In some aspects / applications, the present invention relates to a method for synthesizing antibody-drug conjugates (ADCs) of the present invention.

[0261] In some aspects / modes, the present invention also relates to a method for preparing an antibody conjugate (ADC) of formula (I), the method being: Formula (II) TIFF2026513098000107.tif29128 (in the formula, TIFF2026513098000108.tif6128 is either a triple bond or TIFF2026513098000109.tif6128 is a double bond; If TIFF2026513098000110.tif6128 has a triple bond, then V does not exist, or If TIFF2026513098000111.tif6128 has a double bond, then V is either H or (C1-C8) alkyl; If TIFF2026513098000112.tif6128 has a triple bond, then X is R3-C, or If TIFF2026513098000113.tif7128 has a double bond, then X is The filename is TIFF2026513098000114.tif10128; Y is NR 5 , S, O, or CR 6 R 7 and; R 1 is an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 3 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 4 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 5 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 6 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 7 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; CU stands for connector unit; CM is the cytotoxic part; and m is an integer in the range of 1 to 10. Compounds of, or pharmaceutically acceptable salts or solvates thereof, Formula (III): TIFF2026513098000115.tif13128 (wherein Ab is an anti-TPBG antibody as described herein; and n is an integer in the range of 1 to 20) molecules containing thiols This is a process of causing a reaction, As a result, equation (I) TIFF2026513098000116.tif34128 (in the formula, Ab is the anti-TPBG antibody described herein; In the compound of formula (II) If TIFF2026513098000117.tif6128 has a triple bond, TIFF2026513098000118.tif6128 has a double bond, or In the compound of formula (II) If TIFF2026513098000119.tif6128 has a double bond, TIFF2026513098000120.tif5128 is a single bond; If TIFF2026513098000121.tif6128 is a double bond, then V does not exist, or If TIFF2026513098000122.tif5128 is a single bond, then V is either H or (C1-C8) alkyl; If TIFF2026513098000123.tif6128 is a double bond, then X is R3-C, or If TIFF2026513098000124.tif6128 is a single join, then X is The filename is TIFF2026513098000125.tif9128; Y is NH, S, O, or CH2; R 1 is an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 3is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 4 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 5 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 6 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 7 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; CU stands for connector unit; CM is the cytotoxic part; m is an integer in the range of 1 to 10; and n is an integer in the range of 1 to 20. A process to obtain an antibody-drug conjugate (ADC), or a pharmaceutically acceptable salt or solvate thereof. Includes.

[0262] Preferably, R 3 is H or (C1-C8) alkyl, more preferably R 3 is H. Preferably, R 4 If present, is H or (C1-C8) alkyl; more preferably R 4 If present, it is H. Preferably, R 5 If present, is H or (C1-C8) alkyl; more preferably R 5 If present, it is H. Preferably, R 6 If present, is H or (C1-C8) alkyl; more preferably R 6 If present, it is H. Preferably, R 7 If present, is H or (C1-C8) alkyl; more preferably R 7 If it exists, it is H.

[0263] Preferably, TIFF2026513098000126.tif6128 has a triple bond; V is absent; X is R3-C; and R 3 is H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 3 is H or (C1-C8) alkyl; more preferably, R 3 is H; and TIFF2026513098000127.tif6128 represents a double bond.

[0264] more, TIFF2026513098000128.tif6128 represents a triple bond; V is absent; X represents R3-C, where R3 represents H or (C1~C8) alkyl; and TIFF2026513098000129.tif6128 represents a double bond. Preferably, R3 represents H or (C1-C6) alkyl, more preferably H or (C1-C4) alkyl, and even more preferably H or (C1-C2) alkyl. Even more preferably, R3 is H.

[0265] In some embodiments, TIFF2026513098000130.tif6128 may be a double bond; V is H or (C1~C8) alkyl, preferably V is H; X is TIFF2026513098000131.tif9128; R3 is H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; and TIFF2026513098000132.tif6128 may represent a single bond; more preferably, R 3 is H or (C1-C8) alkyl, more preferably R 3 H is R 4 is H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 4is H or (C1-C8) alkyl, preferably R 4 H is H.

[0266] In some embodiments, TIFF2026513098000133.tif6128 may represent a double bond; V may be H or (C1~C8) alkyl; X is TIFF2026513098000134.tif9128 may be represented; R3 and R4 may independently represent H or (C1~C8) alkyl; and TIFF2026513098000135.tif6128 may represent a single bond. Preferably, R3 and R4 independently represent H or C1-C6 alkyl, more preferably H or C1-C4 alkyl, and even more preferably H or C1-C2 alkyl. Preferably, R3 and R4 are the same; even more preferably, R3, R4, and V are the same. Even more preferably, R3 and R4 are both H. Preferably, V is H or C1-C6 alkyl, more preferably H or C1-C4 alkyl, and even more preferably H or C1-C2 alkyl. Even more preferably, V is H. In a preferred embodiment, R3, R4, and V are each H.

[0267] Figures used in this specification Regarding TIFF2026513098000136.tif8128, it should be noted that each carbon atom is tetravalent, as is generally known to those skilled in the art. Therefore, the structure TIFF2026513098000137.tif18128(wherein X and V are as defined herein, and asterisk( * ) indicates binding to phosphorus) the structure TIFF2026513098000138.tif23128 (wherein R3, R4, and V are as defined herein) It includes structure TIFF2026513098000139.tif18128(wherein X and V are as defined herein, and asterisk( * ) indicates binding to phosphorus, and # indicates binding to receptor-binding molecules (RBMs). The structure This includes TIFF2026513098000140.tif22128 (wherein R3, R4, and V are as defined herein, and H is hydrogen). The wavy bond indicates that the stereochemistry of the double bond can be E or Z. It is also possible for the compound to exist as a mixture of E and Z isomers.

[0268] If the anti-TPBG antibody (Ab) contains one or more disulfide crosslinks, the method may further include a step of reducing at least one disulfide crosslink of the antibody in the presence of a reducing agent to form a thiol group (SH). The resulting compound of formula (III) may then be reacted with the compound of formula (II) to obtain the antibody-drug conjugate (ADC) of formula (I). The reducing agent may be selected from the group consisting of tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), sodium dithionite, sodium thiosulfate, and sodium sulfite. Therefore, the reducing agent may be dithiothreitol (DTT). The reducing agent may be sodium dithionite. The reducing agent may be sodium sulfite. Preferably, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0269] Preferably, the reduction of at least one disulfide bridge involves using about 1 to about 3 equivalents, more preferably about 1 to about 2 equivalents, and even more preferably about 1 equivalent of a reducing agent per disulfide bridge to be reduced. In this context, it should be noted that theoretically, 1 equivalent of a reducing agent, specifically one of the reducing agents described herein, is required to reduce one disulfide bridge to produce two thiol groups (SH).

[0270] Preferably, a molecule containing the thiol of formula (III) is reacted with a compound of formula (II) in an amount of about 1 to about 4 equivalents, preferably about 1 to about 3 equivalents, more preferably about 1 to about 2 equivalents, and even more preferably about 1 to 1.5 equivalents per thiol group (SH).

[0271] Preferably, the reaction between the compound of formula (II) and the molecule containing the thiol of formula (III) is carried out in an aqueous medium.

[0272] Preferably, the reaction between the compound of formula (II) and the molecule containing the thiol of formula (III) is carried out under neutral or slightly basic conditions. More preferably, the reaction is carried out at a pH of 6 to 10. Even more preferably, the reaction is carried out at a pH of 7 to 9.

[0273] In any one of the methods, any variable may be defined as described herein, specifically, as in the case of antibody-drug conjugates (ADCs) of formula (I) and / or compounds of formula (II). Thus, Ab, TIFF2026513098000141.tif9128, V, X, Y, R 1 , R 3 , R 4 , R 5 , R 6 , R 7 CU, CM, m, and n may be as defined herein. Preferably, Y is NH.

[0274] Methods for preparing compounds of formula (II) are known in the art. Exemplary examples include compounds of formula (II) where group Y is NH, which can be prepared by techniques and conditions such as the Staudinger phosphonite reaction described, for example, in WO 2018 / 041985 A1, incorporated herein by reference. Compounds of formula (II) where Y is S or O can be prepared by techniques and conditions described, for example, in WO 2019 / 170710, incorporated herein by reference. Similar to the compounds of formula (II) where Y is S or O described in WO 2019 / 170710, where Y is CR 6 R 7 Compounds of formula (II) can be prepared, as exemplary examples, by substitution of the phosphorus atom, for example, by a suitable organometallic compound such as a Grignard compound or an organolithium compound. Those skilled in the art will readily select appropriate methods and conditions for preparing compounds of formula (II). The examples section of this disclosure also includes guidance on methods for preparing or obtaining compounds of formula (II) and / or antibody-drug conjugates (ADCs) of formula (I).

[0275] The present invention also relates to an antibody-drug conjugate of formula (I) that can be obtained or is obtained by any method for preparing an antibody-drug conjugate of formula (I) as described herein.

[0276] In some aspects / concepts, the present invention relates to cancer. Cancer can be any cancer. Preferably, cancer is selected from the group consisting of breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, cervical cancer, rectal cancer, colon cancer, esophageal cancer, stomach cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcoma, brain cancer, nevus and melanoma, genitourinary cancer, prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancer, retinoblastoma, thyroid cancer, and fallopian tube cancer; more preferably, the cancer is a solid tumor; most preferably, the cancer is breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, uterine cancer (e.g., muscular cancer), cervical cancer, rectal cancer, colon cancer, and anal cancer. The following cancers are selected from the group consisting of esophageal cancer, stomach cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcomas (e.g., osteosarcoma and Kaposi's sarcoma), brain cancer (e.g., pituitary tumors), nevus and melanoma cancer, skin cancer (e.g., squamous cell carcinoma and melanoma), genitourinary cancers (e.g., ureteral and bladder cancer, testicular cancer, prostate cancer, penile cancer), prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancers (e.g., lymphoma, leukemia, myeloma, myelodysplastic syndrome, or myelofibrosis), eye cancers (e.g., retinoblastoma), neuroendocrine tumors, and cancer of unknown primary origin (CUP).

[0277] In some aspects / applications, the present invention relates to compositions or kits comprising anti-TPBG, antibody-drug conjugates (ADCs), hybridomas, nucleic acids, expression vectors, and / or host cells.

[0278] In some aspects / encompassing, the present invention relates to methods of treatment (e.g., of a patient), as well as the use of the antibodies, antibody-drug conjugates (ADCs), nucleic acids, expression vectors, host cells, compositions, and / or kits of the present invention.

[0279] In some aspects of the present invention, the antibody of the present invention is expressed in CHO cells as Fc-silencing (LALA mutation) IgG1 and purified by protein A chromatography.

[0280] In some aspects of the present invention, the mAb of the present invention binds to human 5T4, for example, in a 5T4 / antigen glycosylation-dependent manner, and the apparent dissociation constant (K) in the context of ELISA is D ) is 0.11 nM.

[0281] In some aspects of the present invention, the mAb of the present invention binds to recombinant 5T4-ECD (extracellular domain) and delivers 0.25 nM of K to MDA-MB-468 cells that endogenously express 5T4. D They are joined together.

[0282] In some aspects of the present invention, the mAb of the present invention exhibits cross-reactivity with 5T4 of non-human primates (NHPs), namely marmosets.

[0283] The present invention further relates to the following items: 1. An anti-TPBG (which can also be called anti-5T4) antibody (for example, an antibody against its antigen-binding moiety, preferably TPBG_human trophoblast glycoprotein (e.g., having UniProt accession number: Q13641 or SEQ ID NO: 1)), wherein the anti-TPBG antibody contains Fc silencing mutations such as leucine (L) to alanine (A) substitutions (LALA mutations) at positions 234 and 235 (e.g., in the Fc region of the antibody), and (a) The anti-TPBG antibody is capable of binding to human trophoblast glycoprotein (TPBG) (e.g., having UniProt accession number: Q13641 or SEQ ID NO: 1); (b) The anti-TPBG antibody may cross-react with marmoset TPBG (e.g., common marmoset) which has white tufts of hair around its ears (e.g., has UniProtKB accession number: F7I0T3 or SEQ ID NO: 2); and (c) The anti-TPBG antibody is capable of internal migration, preferably by internal antibody migration via the antigen. Anti-TPBG antibody. 2. A mutated Fc region (or crystalline fragment region) (according to any one of the above items), for example, the tail region of the antibody that interacts with the cell surface Fc receptor in the unmutated state (for example, where the Fc region of the immunoglobulin molecule consists of a constant region of the heavy chain, which, for example, can bind to the antibody receptor (Fc receptor) on the cell and the C1q component of complement in the unmutated state). An anti-TPBG antibody containing any one of the items mentioned above. 3. An anti-TPBG antibody containing a heavy chain constant region, one of the above items. 4. An anti-TPBG antibody that can bind (e.g., specifically bind) to the extracellular domain of TPBG (e.g., amino acids 32-355 of SEQ ID NO: 1), any one of the above items. 5. An anti-TPBG antibody that can bind (e.g., specifically bind) to any one of the above items, to the extracellular epitope of TPBG (e.g., amino acids 32-355 of SEQ ID NO: 1). 6. (a) The anti-TPBG antibody, (for example, in MDA-MB-468 cells that endogenously express TPBG (e.g., having DSMZ number: ACC738)), has a K content in the range of about 0.01 to about 10 nmol / L, preferably in the range of about 0.15 to about 0.35 nmol / L, relative to endogenously expressed (e.g., expressed on the cell surface) human TPBG. D Having, and more preferably the K D This is measured by the FACS assay method, and the anti-TPBG antibody is most preferably in the range of about 0.20 to about 0.30 nmol / L (e.g., 0.25 nmol / L) D Having; and / or (b) The anti-TPBG antibody is optionally present in a K2 concentration of approximately 0.01 to approximately 10 nmol / L against immobilized exogenous full-length TPBG and / or one or more fragments thereof. DThe fragments include, preferably, at least one extracellular domain (ECD) of the TPBG (e.g., the ECD contains at least 32-355 amino acids of human TPBG having UniProt accession number: Q13641 or SEQ ID NO: 1) and / or one or more fragments of the ECD (e.g., having a length of about 15-30 amino acids), and the full-length TPBG and / or one or more fragments thereof are fused to or not fused to one or more protein tags (e.g., 6×His tag, FLAG, HA, V5, Fc fusion, MBP, SUMO, TEV, GFP, TST), and more preferably, the K D The range is approximately 0.05 to approximately 0.30 nmol / L, and most preferably, the K D This is measured by an ELISA assay, and more preferably, the K D The range is approximately 0.05 to 0.2 nmol / L (for example, approximately 0.10 nmol / L (e.g., glycosylated) to approximately 0.16 nmol / L (deglycosylated)). One of the anti-TPBG antibodies listed above. 7. The anti-TPBG antibody has a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 3, and SEQ ID NO: An antibody comprising a light chain variable region having an amino acid sequence that has at least 80% identity with respect to 4 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), Preferably, the anti-TPBG antibody is (a) A light chain containing an amino acid sequence having at least 80% identity with SEQ ID NO: 5 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), (b) SEQ ID NO: A heavy chain comprising an amino acid sequence having at least 80% identity to 6 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) One of the anti-TPBG antibodies listed above. 8. Anti-TPBG antibodies, (a) A heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 7, heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 8, and heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 9, (b) An anti-TPBG antibody that includes a light chain variable region comprising a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 10, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 11, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 12, which is any one of the above items. 9. The following characteristics: (a) Monoclonal antibody; (b) Chimeric antibodies and / or humanized antibodies; (c) Specific recognition of (e.g., human) TPBG that is overexpressed in cancer cells; (d) Human IgG antibody, preferably human IgG1 antibody; (e) Contains kappa (κ) light chain; (f) Contains a lambda (λ) light chain; (g) It can be internally transported by target cells expressing TPBG (e.g., cancer cells). (Preferably, the internally transported antibody is directed towards the lysosome); (h) Tumor-selective antibodies (Preferably, the tumor is a liquid tumor and / or a solid tumor, preferably a solid tumor); (i) Antibodies selective for malignant cells; (j) Binding to human TPBG in a glycosylation-dependent manner. (Here, preferably, the antibody binds to the glycosylated form of the TPBG); (k) Includes Fc silencing mutations such as leucine (L) to alanine (A) substitutions at positions 234 and 235 (LALA mutations, i.e., Leu234Ala and Leu235Ala). (Here, the LALA mutation can reduce the effector function of immune cells.) An anti-TPBG antibody having one or more of the above items. 10. A monoclonal anti-TPBG antibody, one of the above-mentioned antibodies. 11. A chimeric anti-TPBG antibody, which is one of the anti-TPBG antibodies listed above. 12. A humanized anti-TPBG antibody, one of the above-mentioned antibodies. 13. An anti-TPBG antibody that specifically recognizes (e.g., human) TPBG, which is overexpressed in cancer cells, and any one of the above items. 14. An anti-TPBG antibody, preferably a human IgG antibody, which is any one of the above items. 15. A human IgG1 antibody, which is an anti-TPBG antibody for any one of the items listed above. 16. An anti-TPBG antibody containing a kappa (κ) light chain, one of the above items. 17. An anti-TPBG antibody containing a lambda (λ) light chain, one of the above items. 18. An anti-TPBG antibody can be internally transported by target cells expressing TPBG (e.g., cancer cells); preferably, the internally transported antibody is directed towards lysosomes, one of the anti-TPBG antibodies described above. 19. An anti-TPBG antibody that is tumor-selective, preferably the tumor is a liquid tumor and / or a solid tumor, preferably a solid tumor, any one of the above items. 20. An anti-TPBG antibody that is selective for malignant cells, one of the above-mentioned antibodies. 21. An anti-TPBG antibody that can bind to human TPBG in a glycosylation-dependent manner, and preferably to glycosylated TPBG, any one of the above items. 22. An anti-TPBG antibody comprising an Fc silencing mutation, such as a leucine (L) to alanine (A) substitution (LALA mutation) at positions 234 and 235, wherein the LALA mutation can reduce the effector function of immune cells, wherein the anti-TPBG antibody comprises any one of the above items. 23. An anti-TPBG antibody, one of the above items, bound to a labeling group. 24. An anti-TPBG antibody from any of the above items, which can be obtained using a hybridoma (for example, a recombinant antibody). 25. An anti-TPBG antibody comprising one or more signal sequences described in SEQ ID NO: 13 and / or SEQ ID NO: 14, any one of the above items. 26. Preferably selected from the group consisting of SEQ ID NO: 3 to 14, one or more anti-TPBG antibodies comprising any one of the above items, including CDRs (e.g., 2, 3, 4, 5, or 6), heavy chain variable regions (e.g., 2), light chain variable regions (e.g., 2), heavy chains (e.g., 2), light chains (e.g., 2), and / or signal sequences. 27. An anti-TPBG antibody of any one of the above items, obtained according to Example 1, 2, or 3 of this Specified Specified, and / or having the features described in Example 1, 2, or 3 of this Specified 28. Consists of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions (or mutations), which are preferably: CDR (e.g., CDR1, CDR2, CDR3, e.g., CDR-H1, CDR-H2, CDR-H3, CDR-H1, CDR-L1, CDR-L2, and / or CDR-L3, e.g., those listed in any one of the above items, e.g., the sequence listings disclosed herein), V H (Variable region heavy chain), V L (Variable region light chain), C H (Steady region heavy chain), or C L An anti-TPBG antibody of any one of the items, located within one or more regions selected from the group consisting of (constant region light chain), signal sequence, F(ab), and / or Fc region (for example, as defined in Figure 1 herein). 29. Containing one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) highly conserved, conserved, or equivalent amino acid substitutions (or mutations), For example, “conservative or equivalent substitutions” means the substitutions listed as “exemplary substitutions” in Table I below, and “highly conservative substitutions” means, as used herein, the substitutions shown under the heading “preferred substitutions” in Table I below. (Table I) Amino Acid Substitutions Preferably, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) highly conserved, conserved, or equivalent amino acid substitutions (or mutations) are: CDR (e.g., CDR1, CDR2, CDR3, e.g., CDR-H1, CDR-H2, CDR-H3, CDR-H1, CDR-L1, CDR-L2, and / or CDR-L3, e.g., those listed in any one of the above items, e.g., the sequence listings disclosed herein), V H (Variable region heavy chain), V L (Variable region light chain), C H (Steady region heavy chain), or C LAn anti-TPBG antibody of any one of the items, located within one or more regions selected from the group consisting of (constant region light chain), F(ab), and / or the Fc region (for example, as defined in Figure 21 herein). 30. A hybridoma that produces any one of the monoclonal antibodies listed above. 31. A nucleic acid encoding any one of the antibodies listed above. 32. An expression vector comprising at least one nucleic acid molecule from any of the items described above. 33. An isolated host cell (e.g., an isolated recombinant host cell) containing any one of the vectors and / or nucleic acids mentioned above. 34. An antibody-drug conjugate (ADC) comprising any one of the above anti-TPBG antibodies (for example, obtained according to Example 1, 2, or 3 of this Specified Specified, and / or having the features described in Example 1 and / or Example 2 and / or Example 3 of this Specified Specified). 35. The anti-TPBG antibody has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) cytotoxic moieties (e.g., cytotoxic payload, e.g., tubes) An antibody-drug conjugate (ADC) of any one of the above items, conjugated to a brin inhibitor, such as a topoisomerase I inhibitor, such as auristatin or camptothecin, such as MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), such as exatecan (e.g., CAS number: 171335-80-1), preferably via one or more linkers, more preferably via one or more phosphoneamidate linkers. 36. An antibody-drug conjugate (ADC) of any one of the above items, wherein an anti-TPBG antibody is conjugated to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) camptothecin (e.g., exatecan) cytotoxic moieties, preferably via one or more linkers, more preferably via one or more phosphoamidate linkers. 37. An antibody-drug conjugate (ADC) of any one of the above items, wherein an anti-TPBG antibody is conjugated to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) exatecan cytotoxic moieties via one or more linkers, preferably via one or more phosphoamidate linkers. 38. The antibody-drug conjugate (ADC) contains a humanized monoclonal TPBG-specific IgG1 antibody conjugated to a cytotoxic payload. (a) The cytotoxic payload is selected from the group consisting of camptothecin, mytansinoids, calicheamycin, duocalmycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and their analogues or prodrugs; and / or (b) The cytotoxic payload is a camptothecin moiety C selected from the group consisting of exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, berotecan, lulutotecan, rubitecan, siratecan, cocitecan, and gimatecan; and / or (c) The cytotoxic payload is conjugated via a cleavable linker (L), preferably the linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction, and more preferably the linker is cleavable by a protease, preferably a cathepsin such as cathepsin B; and / or (d) The linker L comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) The cytotoxic payload is an exatecan conjugated via a chemical valine-citrulline-PAB free unit or a valine-alanine-PAB free unit, wherein the free unit is cleavable by a protease. An antibody-drug conjugate (ADC) from any of the above items. 39. The anti-TPBG antibody is present in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) camptothecin (e.g., exatecan) cytotoxic moieties. For example, the antibody-drug conjugate (ADC) is conjugated via conjugation by an ethinylphosphoneamidate linker (to all eight interchain cysteine ​​residues); preferably, each phosphoneamidate linker has at least one PEG24 portion (for example, to prevent aggregation of the antibody-drug conjugate (ADC)); and more preferably, the ADC has up to eight linker payload portions and eight PEG24 portions, any one of the above items. 40. Equation (I): Having TIFF2026513098000143.tif34128 or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, Ab is one of the anti-TPBG antibodies listed above; TIFF2026513098000144.tif6128 is either a double bond or TIFF2026513098000145.tif6128 is a single bond; If TIFF2026513098000146.tif6128 has a double bond, then V does not exist, or If TIFF2026513098000147.tif6128 is a single bond, then V is either H or (C1-C8) alkyl; If TIFF2026513098000148.tif6128 is a double bond, then X is R3-C, or If TIFF2026513098000149.tif6128 is a single join, then X is The filename is TIFF2026513098000150.tif9128; Y is NR 5 , S, O, or CR 6 R 7 and; R 1 is an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 3 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 4 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 5 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 6 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 7 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; CU stands for connector unit; CM is the cytotoxic part; m is an integer in the range of 1 to 10; and n is an integer in the range of 1 to 20. Optionally, one antibody-drug conjugate (ADC) from items 34-39. 41. R 3 is H or (C1-C8) alkyl; preferably, R 3 H is; R 4 However, if present, it is H or (C1-C8) alkyl; preferably, R 4 However, if it exists, it is H; R 5 However, if present, it is H or (C1-C8) alkyl; preferably, R 5 However, if it exists, it is H; R 6However, if present, it is H or (C1-C8) alkyl; preferably, R 6 However, if it exists, it is H; and R 7 However, if present, it is H or (C1-C8) alkyl; preferably, R 7 However, if it exists, it is H. Item 40: Antibody-drug conjugates (ADCs). 42. TIFF2026513098000151.tif6128 has a double bond; V is absent; X is R3-C; and R 3 However, H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 3 is H or (C1-C8) alkyl; more preferably R 3 An antibody-drug conjugate (ADC) of item 40 or 41, where H is present. 43. TIFF2026513098000152.tif6128 is a single bond; V is H or (C1~C8) alkyl, preferably V is H; X is TIFF2026513098000153.tif9128 is;R 3 However, H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; more preferably, R 3 is H or (C1-C8) alkyl, more preferably R 3 H is R 4 However, H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 4 However, it is H or (C1-C8) alkyl, preferably R 4 An antibody-drug conjugate (ADC) of item 40 or 41, where H is present. 44. One antibody-drug conjugate (ADC) from items 40-43, where Y is NH. 45. One antibody-drug conjugate (ADC) from items 40-44, with a connector unit (CU) that is detachable. 46. ​​Antibody-drug conjugates (ADCs) of item 45 in which the connector unit CU can be cleaved by protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction. 47. An antibody-drug conjugate (ADC) of item 46, wherein the connector unit CU is cleavable by a protease, preferably a cathepsin, such as cathepsin B. 48. An antibody-drug conjugate (ADC) from any of items 40-47, in which the connector unit (CU) contains a valine-citrulline moiety. 49. Connector unit CU, The filename is TIFF2026513098000154.tif50128. In the formula, # indicates a connection point to Y. * This indicates a binding site to the cytotoxic portion CM. Item 48: Antibody-drug conjugates (ADCs). 50. The connector unit CU is one of the antibody-drug conjugates (ADCs) from items 40-47, which contains a valine-alanine moiety. 51. Connector unit CU, The filename is TIFF2026513098000155.tif37128. During the ceremony, * indicates a binding point to Y, and ## indicates a binding point to the cytotoxic region. Item 50 antibody-drug conjugates (ADCs). 52. An antibody-drug conjugate (ADC) from any of items 40-44 whose linker is not cleavable. 53. R 1 However, it is a polyethylene glycol unit, one of the antibody-drug conjugates (ADCs) listed in items 40-52. 53a. Polyethylene glycol units, structure: Antibody-drug conjugates (ADCs) of item 53, containing 1 to 100 subunits having TIFF2026513098000156.tif9128. 54. R 1 but, The filename is TIFF2026513098000157.tif15128. During the ceremony, TIFF2026513098000158.tif9128 indicates the position of O; K F -H, -PO3H, -(C1~C 10 )alkyl, -(C1~C 10 )alkyl-SO3H,-(C2~C 10 )alkyl-CO2H,-(C2~C 10 )alkyl-OH, -(C2~C 10 )alkyl-NH2,-(C2~C 10 )alkyl-NH(C1~C3)alkyl, and -(C2~C 10 Selected from the group consisting of alkyl-N((C1~C3)alkyl)2; and o is an integer in the range of 1 to 100. One antibody-drug conjugate (ADC) from any of items 40-53a. 55. K F The antibody-drug conjugate (ADC) of item 54 is H. 56. Antibody-drug conjugates (ADCs) of item 54 or 55, where o is in the range of 8-30. 57. Antibody-drug conjugates (ADCs) of item 56, where o is in the range of 20-28. 58. An antibody-drug conjugate (ADC) of item 57, where o is 22, 23, 24, 25, or 26. 59. Antibody-drug conjugates (ADCs) of item 56, where o is in the range of 8-16. 60. Antibody-drug conjugates (ADCs) of item 59, where o is 10, 11, 12, 13, or 14. 61. An antibody-drug conjugate (ADC) from any one of items 40-60, wherein the cytotoxic portion CM is selected from the group consisting of camptothecin, mytansinoids, calicheamycin, duochamycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and their analogs or prodrugs. 62. An antibody-drug conjugate (ADC) of item 61 in which the cytotoxic moiety (CM) is the camptothecin moiety. 63. An antibody-drug conjugate (ADC) of item 62 in which the camptothecin portion is selected from the group consisting of exatecan, DXD, SN38, camptothecan, topotecan, irinotecan, berotecan, lulutotecan, rubitecan, silatecan, cocitecan, and gimatecan. 64. The cytotoxic part CM is, formula: Exatecan, item 63, is an antibody-drug conjugate (ADC) with the code TIFF2026513098000159.tif56128. 65. The cytotoxic part CM is, formula: Exatecan, item 64, is an antibody-drug conjugate (ADC) with the code TIFF2026513098000160.tif52128. 66. An antibody-drug conjugate (ADC) of item 64 or 65, in which exatecan is bound to the connector unit CU via an amino group. 67. Any one antibody-drug conjugate (ADC) from item 40 to 66, wherein the number of cytotoxic moieties (CMs) per receptor-binding molecule, particularly the number of camptothecin moieties, is 1 to 14, preferably 2 to 14, more preferably 4 to 14, even more preferably 5 to 12, even more preferably 6 to 12, even more preferably 7 to 10, and still more preferably 8. 68. Any one of items 40-66, an antibody-drug conjugate (ADC) in which the number of cytotoxic moieties (CMs) per receptor-binding molecule, particularly the number of camptothecin moieties, is 1 to 14, preferably 1 to 12, more preferably 2 to 10, even more preferably 2 to 8, even more preferably 2 to 6, even more preferably 3 to 5, and still more preferably 4. 69. Any one antibody-drug conjugate (ADC) from items 40 to 66, where m is an integer in the range of 1 to 4, preferably 1 or 2, more preferably 1; and n is an integer in the range of 1 to 20, preferably 1 to 10, more preferably 2 to 10, even more preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, and even more preferably 8. 70. An antibody-drug conjugate of any one of items 40-66, where m is an integer in the range of 1 to 4, preferably 1 or 2, more preferably 1; and n is an integer in the range of 1 to 20, preferably 1 to 10, more preferably 2 to 8, even more preferably 3 to 6, even more preferably 4 or 5, and still more preferably 4. 71. Equation (I): Having TIFF2026513098000161.tif34128 or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, Ab is one of the anti-TPBG antibodies listed above; TIFF2026513098000162.tif6128 is either a double bond or TIFF2026513098000163.tif6128 is a single bond; If TIFF2026513098000164.tif6128 has a double bond, then V does not exist, or If TIFF2026513098000165.tif6128 is a single bond, then V is H; If TIFF2026513098000166.tif6128 has a double bond, then X is R3-C, or If TIFF2026513098000167.tif6128 is a single join, then X is The filename is TIFF2026513098000168.tif9128; Y is NH; R 1 Structure: This is a polyethylene glycol unit having TIFF2026513098000169.tif15128. During the ceremony, TIFF2026513098000170.tif10128 indicates the position of O, K F is H and o is an integer in the range of 8 to 30; R 3 is H; R 4 is H; The CU has the following structure: This is a connector unit having TIFF2026513098000171.tif52128, where # indicates the connection point to Y. * This indicates the binding site to the camptothecin moiety (CM); CM is the camptothecin part; m is 1; and n is an integer in the range of 1 to 10. Optionally, one antibody-drug conjugate (ADC) from item 40 to 70. 72. TIFF2026513098000172.tif6128 has a double bond; V is absent; X is R3-C; and R 3 The antibody-drug conjugate (ADC) of item 71 is H. 72a. TIFF2026513098000173.tif6128 is a single bond; V is H; X is TIFF2026513098000174.tif9128 is;R 3 H is and R 4 The antibody-drug conjugate (ADC) of item 71 is H. 73. The camptothecin portion CM is given by formula: An antibody-drug conjugate (ADC) of any one of items 71-72a, which is exatecan having TIFF2026513098000175.tif53128. 74. An antibody-drug conjugate (ADC) of item 73, in which exatecan is bound to the connector unit CU via an amino group. 75. One antibody-drug conjugate (ADC) from items 71-74, where o is in the range of 20-28. 76. An antibody-drug conjugate (ADC) of item 75, where o is 22, 23, 24, 25, or 26. 77. One antibody-drug conjugate (ADC) from items 71-76, where n is in the range of 2-10. 78. Antibody-drug conjugates (ADCs) of item 77 where n is 8. 79. Antibody-drug conjugates (ADCs) of item 77 where n is 4. 80. The following equation (Ia): It has TIFF2026513098000176.tif67157, In the formula, Ab is one of the anti-TPBG antibodies listed above. Optionally, one of the antibody-drug conjugates (ADCs) from items 71-78. 81. An antibody-drug conjugate (ADC) of item 61 in which the cytotoxic portion (CM) is auristatin. 82. An antibody-drug conjugate (ADC) of item 81, wherein the cytotoxic moiety is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). 83. An antibody-drug conjugate (ADC) of item 82, wherein the cytotoxic moiety is monomethyl auristatin E (MMAE). 84. Antibody-drug conjugates (ADCs) are as follows: a) Equation I: TIFF2026513098000177.tif56137b) Formula II: In formula TIFF2026513098000178.tif46128, n is in the range of 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8); c) Formula III: In formula TIFF2026513098000179.tif42128, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); d) Formula IV: In formula TIFF2026513098000180.tif41128, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); e) Formula V: In formula TIFF2026513098000181.tif43128, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); f) Equation VI: In formula TIFF2026513098000182.tif45128, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); g) Formula VII: In formula TIFF2026513098000183.tif41128, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); h) Formula VIII: In formula TIFF2026513098000184.tif41128, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); i) Formula IX: In formula TIFF2026513098000185.tif49128, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); j) Formula X: In formula TIFF2026513098000186.tif41128, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); k) Formula XI: In formula TIFF2026513098000187.tif43128, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); l) Formula XII: In formula TIFF2026513098000188.tif44132, n is in the range of 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8); m) Formula XIII: In formula TIFF2026513098000189.tif48131, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); n) Formula XIV: In formula TIFF2026513098000190.tif45133, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); o) Formula XV: In formula TIFF2026513098000191.tif45135, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); p) Formula XVI: In formula TIFF2026513098000192.tif51139, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); The formula is selected from the group consisting of, Here, TIFF2026513098000193.tif32128 is one of the anti-TPBG antibodies listed above. An antibody-drug conjugate (ADC) from any of the above items. 85. a) Anti-TPBG monoclonal antibodies can specifically recognize (e.g., human) TPBG overexpressed in cancer cells; and b) When an antibody-drug conjugate (ADC) binds to TPBG overexpressed in cancer cells, the ADC can be internalized by the cell and transported into the lysosomal compartment, where preferably a lysosomal protease (e.g., cathepsin B) can release a cytotoxic payload from the ADC. An antibody-drug conjugate (ADC) from any of the above items. 86. Formula (II): Compounds containing TIFF2026513098000194.tif29128, or pharmaceutically acceptable salts or solvates thereof. During the ceremony, TIFF2026513098000195.tif6128 is either a triple bond or TIFF2026513098000196.tif6128 is a double bond; If TIFF2026513098000197.tif6128 has a triple bond, then V does not exist, or If TIFF2026513098000198.tif6128 has a double bond, then V is either H or (C1-C8) alkyl; If TIFF2026513098000199.tif6128 has a triple bond, then X is R3-C, or If TIFF2026513098000200.tif6128 has a double bond, then X is The filename is TIFF2026513098000201.tif9128; Y is NR 5 , S, O, or CR 6 R 7 and; R 1 is an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 3 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 4 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 5 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 6 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 7 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; CU stands for connector unit; CM is the cytotoxic part; and m is an integer in the range of 1 to 10. 87. R 3 is H or (C1-C8) alkyl; preferably, R 3 H is; R 4 However, if present, it is H or (C1-C8) alkyl; preferably, R 4 However, if it exists, it is H; R 5 However, if present, it is H or (C1-C8) alkyl; preferably, R 5 However, if it exists, it is H; R 6 However, if present, it is H or (C1-C8) alkyl; preferably, R 6 However, if it exists, it is H; and R 7 However, if present, it is H or (C1-C8) alkyl; preferably, R 7 However, if it exists, it is H. Compounds of item 86. 88. TIFF2026513098000202.tif6128 has a triple bond; V is absent; X is R3-C; and R 3 However, H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 3 is H or (C1-C8) alkyl; more preferably R 3 Compounds of item 86 or 87 in which is H. 89. TIFF2026513098000203.tif6128 is a double bond; V is H or (C1~C8) alkyl, preferably V is H; X is TIFF2026513098000204.tif9128 is;R 3 However, H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 3is H or (C1-C8) alkyl, more preferably R 3 H is R 4 However, H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 4 is H or (C1-C8) alkyl, more preferably R 4 Compounds of item 86 or 87 in which is H. 90. Ab, V, X, Y, R 1 , R 3 , R 4 , R 5 , R 6 , R 7 ,CU,CM,m, and n are as defined in any one of items 40 to 85; preferably, one of the compounds in items 86 to 89, where Y is NH. 91. (a) Conjugate one of the antibodies from the above items to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) cytotoxic moieties (e.g., cytotoxic payloads, e.g., tubulin inhibitors, e.g., topoisomerase I inhibitors, e.g., auristatin or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., exatecan) preferably via one or more linkers, more preferably via one or more phosphoamidate linkers. A method for producing antibody-drug conjugates (ADCs) containing [the specified substance]. 92. A method for producing an antibody-drug conjugate (ADC) of any one of the above items, wherein an anti-TPBG antibody is conjugated to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) camptothecin (e.g., exatecan) cytotoxic moieties, preferably via one or more linkers, more preferably via one or more phosphoamidate linkers. 93. A method for producing an antibody-drug conjugate (ADC) of any one of the above items, wherein an anti-TPBG antibody is conjugated to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) exatecan cytotoxic moieties via one or more linkers, preferably one or more phosphoamidate linkers. 94. The anti-TPBG antibody is present in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) camptothecin (e.g., exatecan) cytotoxic moieties (e.g., all 8) A method for producing an antibody-drug conjugate (ADC) of any one of the above items, conjugated via conjugation by an ethinylphosphoneamidate linker to an interchain cysteine ​​residue; preferably, each phosphoneamidate linker has at least one PEG portion having up to 24 PEG units (for example, to prevent aggregation of the antibody-drug conjugate (ADC)), and more preferably, the ADC has up to 8 linker payload portions and 8 PEG24 portions. 95. A method for producing one of the antibody-drug conjugates (ADCs) described above, The ADC comprises a humanized monoclonal TPBG-specific IgG1 antibody conjugated to a cytotoxic payload. (a) The cytotoxic payload is selected from the group consisting of camptothecin, mytansinoids, calicheamycin, duocalmycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and their analogues or prodrugs; and / or (b) The cytotoxic payload is a camptothecin moiety C selected from the group consisting of exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, berotecan, lulutotecan, rubitecan, siratecan, cocitecan, and gimatecan; and / or (c) The cytotoxic payload is conjugated via a cleavable linker (L), preferably the linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction, and more preferably the linker is cleavable by a protease, preferably a cathepsin such as cathepsin B; and / or (d) The linker L comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) The cytotoxic payload is an exatecan conjugated via a chemical valine-citrulline-PAB free unit or a valine-alanine-PAB free unit, wherein the free unit is cleavable by a protease. The method. 96. The following steps: Formula (II) TIFF2026513098000205.tif29128 (in the formula, TIFF2026513098000206.tif6128 is either a triple bond or TIFF2026513098000207.tif6128 is a double bond; If TIFF2026513098000208.tif6128 has a triple bond, then V does not exist, or If TIFF2026513098000209.tif6128 has a double bond, then V is either H or (C1-C8) alkyl; If TIFF2026513098000210.tif6128 has a triple bond, then X is R3-C, or If TIFF2026513098000211.tif6128 has a double bond, then X is The filename is TIFF2026513098000212.tif9128; Y is NR 5 , S, O, or CR 6 R 7 and; R 1is an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 3 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 4 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 5 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 6 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 7 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; CU stands for connector unit; CM is the cytotoxic part; and m is an integer in the range of 1 to 10. The compound or a pharmaceutically acceptable salt or solvate thereof Formula (III): TIFF2026513098000213.tif13128 (wherein Ab is one of the anti-TPBG antibodies listed above; n is an integer in the range of 1 to 20) molecules containing thiols This is a process of causing a reaction, As a result, equation (I) TIFF2026513098000214.tif34128 (in the formula, Ab is one of the anti-TPBG antibodies listed above; In the compound of formula (II) If TIFF2026513098000215.tif6128 has a triple bond, TIFF2026513098000216.tif6128 has a double bond, or In the compound of formula (II) If TIFF2026513098000217.tif6128 has a double bond, TIFF2026513098000218.tif6128 is a single bond; If TIFF2026513098000219.tif6128 has a double bond, then V does not exist, or If TIFF2026513098000220.tif6128 is a single bond, then V is either H or (C1-C8) alkyl; If TIFF2026513098000221.tif6128 is a double bond, then X is R3-C, or If TIFF2026513098000222.tif6128 is a single join, then X is The filename is TIFF2026513098000223.tif9128; Y is NR 5 , S, O, or CR 6 R 7 and; R 1 is an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 3 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 4 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 5 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 6 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 7 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; CU stands for connector unit; CM is the cytotoxic part; m is an integer in the range of 1 to 10; and n is an integer in the range of 1 to 20. A process to obtain an antibody-drug conjugate (ADC), or a pharmaceutically acceptable salt or solvate thereof. A method for preparing an antibody-drug conjugate (ADC) that includes, optionally, one of items 91-95. 97. R 3 is H or (C1-C8) alkyl; preferably, R 3 H is; R 4 However, if present, it is H or (C1-C8) alkyl; preferably, R 4 However, if it exists, it is H; R 5 However, if present, it is H or (C1-C8) alkyl; preferably, R 5 However, if it exists, it is H; R 6 However, if present, it is H or (C1-C8) alkyl; preferably, R 6 However, if it exists, it is H; and R 7 However, if present, it is H or (C1-C8) alkyl; preferably, R 7 However, if it exists, it is H. Method 96. 98. TIFF2026513098000224.tif6128 has a triple bond; V is absent; X is R3-C; R 3 However, H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue, preferably R 3 is H or (C1-C8) alkyl, more preferably R 3 H is and TIFF2026513098000225.tif6128 is a double bond, as per item 96 or 97. 99. TIFF2026513098000226.tif6128 is a double bond; V is H or (C1~C8) alkyl, preferably V is H; X is TIFF2026513098000227.tif9128 is;R 3 However, H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 3 is H or (C1-C8) alkyl, more preferably R 3 H is R 4 However, it is H or (C1-C8) alkyl, preferably R 4 H is and TIFF2026513098000228.tif6128 is a single join, according to item 96 or 97. 100. One of the methods of items 96-99, wherein the reaction is carried out under neutral or slightly basic conditions, preferably at pH 6-10. 101. Any one of items 96 to 100, further comprising the step of reducing at least one disulfide crosslink of an antibody in the presence of a reducing agent, thereby forming a thiol group (SH). 102. The reducing agent is selected from the group consisting of tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), sodium dithionite, sodium thiosulfate, and sodium sulfite; Preferably, the method of item 101, wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP). 103. The method of item 101 or 102, wherein the step of reducing at least one disulfide bridge comprises using about 1 to about 3 equivalents, preferably about 1 to about 2 equivalents, more preferably about 1 equivalent of a reducing agent per disulfide bridge to be reduced. 104. One of the methods described in items 96 to 103, wherein a molecule containing a thiol of formula (III) is reacted with a compound of formula (II) in an amount of about 1 to about 4 equivalents, preferably about 1 to about 3 equivalents, more preferably about 1 to about 2 equivalents, and even more preferably about 1.5 equivalents per thiol group (SH). 105. One of the methods described in items 96-104, wherein the step of reacting a compound of formula (II) with a molecule containing a thiol of formula (III) is carried out in an aqueous medium. 106. Ab, V, X, Y, R 1 , R 3 , R 4 , R 5 , R 6 , R 7 , CU, CM, m, and n are as defined in any one of items 40 to 85; preferably, Y is NH2, in any one of items 96 to 105. 107. An antibody-drug conjugate (ADC) produced by any one of the methods described above. 108. Any one of the above-mentioned antibody-drug conjugates (ADCs) having a drug-to-antibody ratio (DAR) in the range of 0 to 20, preferably in the range of 1 to 20, more preferably in the range of 2 to 12, most preferably in the range of 4 to 10, and most preferably in the range of 4 to 8. 109. An antibody-drug conjugate (ADC) having a DAR of 4 or 8, preferably 8, which is one of the above items. 110. A composition or kit comprising any one of the items described above: anti-TPBG, antibody-drug conjugate (ADC), hybridoma, nucleic acid, expression vector, and / or host cell. 111. Any one of the above items, which is a pharmaceutical and / or diagnostic composition. 112. Any one of the above compositions or kits, wherein the ratio (DAR) of a drug (e.g., cytotoxic moiety (e.g., cytotoxic payload, e.g., tubulin inhibitor, e.g., topoisomerase I inhibitor, e.g., auristatin or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., exatecan) to an antibody is in the range of 0 to 20, preferably in the range of 1 to 20, more preferably in the range of 2 to 12, most preferably in the range of 4 to 10, and most preferably in the range of 4 to 8 (e.g., 4 or 8). 113. Methods for treating, improving, preventing and / or diagnosing cancer, (for example, as in Example 2 or Example 3 of this Spec.), preferably, the cancer being breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, cervical cancer, rectal cancer, colon cancer, esophageal cancer, stomach cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcoma, brain cancer, nevus and melanoma, genitourinary cancer, prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, The cancer is selected from the group consisting of endocrine cancer, thoracic cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancer, retinoblastoma, thyroid cancer, and fallopian tube cancer; more preferably, the cancer is a solid tumor; most preferably, the cancer is breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, uterine cancer (e.g., muscular cancer), cervical cancer, rectal cancer, colon cancer, anal cancer, esophageal cancer, stomach cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, and liver cancer. Organ cancers, sarcomas (e.g., osteosarcoma and Kaposi's sarcoma), brain cancers (e.g., pituitary tumors), nevus and melanoma cancers, skin cancers (e.g., squamous cell carcinoma and melanoma), genitourinary cancers (e.g., ureteral and bladder cancers, testicular cancers, prostate cancers, penile cancers), prostate cancers, vulvar squamous cell carcinomas, oropharyngeal cancers, endocrine cancers, chest cancers, mesothelioma, pancreatic cancers, bile duct cancers, hematological cancers (e.g., lymphoma, leukemia, myeloma, myelodysplastic syndromes, A method selected from the group consisting of (or myelofibrosis), eye cancer (e.g., retinoblastoma), neuroendocrine tumors, cancer of unknown primary origin (CUP), breast cancer, colon cancer, endometrial cancer, sarcoma, gastric cancer, head and neck cancer, pancreatic cancer, lung cancer, mesothelioma, and ovarian cancer, comprising the step of administering a therapeutically effective or prophylactic amount of one of the aforementioned antibodies, antibody-drug conjugates (ADCs), nucleic acids, expression vectors, host cells, compositions, or kits. The method. 114. Any antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit for use as a pharmaceutical and / or in a therapeutic setting. 115. The following methods: (a) A method for treating, improving, preventing and / or diagnosing cancer, wherein the cancer is selected from the group consisting of breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, cervical cancer, rectal cancer, colon cancer, esophageal cancer, gastric cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcoma, brain cancer, nevus and melanoma, genitourinary cancer, prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, cholangiocarcinoma, hematological cancer, retinoblastoma, thyroid cancer, and fallopian tube cancer; more preferably, the cancer is a solid tumor; most preferably, the cancer is breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, uterine cancer (e.g., muscular cancer), cervical cancer, rectal cancer, A method selected from the group consisting of colon cancer, anal cancer, esophageal cancer, stomach cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcoma (e.g., osteosarcoma and Kaposi's sarcoma), brain cancer (e.g., pituitary tumor), nevus and melanoma cancer, skin cancer (e.g., squamous cell carcinoma and melanoma), genitourinary cancer (e.g., ureteral cancer and bladder cancer, testicular cancer, prostate cancer, penile cancer), prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancer (e.g., lymphoma, leukemia, myeloma, myelodysplastic syndrome, or myelofibrosis), eye cancer (e.g., retinoblastoma), neuroendocrine tumor, cancer of unknown primary origin (CUP), breast cancer, colon cancer, endometrial cancer, sarcoma, stomach cancer, head and neck cancer, pancreatic cancer, lung cancer, mesothelioma, and ovarian cancer; (b) Methods for monitoring the progression of cancer, and / or for evaluating the effectiveness and / or toxicity of cancer treatments or anticancer compounds / agents; (c) Methods for screening candidate compounds for anticancer activity; (d) Methods for altering the resistance of cancer cells to chemotherapy; (e) Methods for making cancer cells sensitive to chemotherapy; (f) Methods for inhibiting the proliferation of cancer cells expressing TPBG; (g) Methods for the production or preparation of antibodies; (h) Methods for immunizing non-human animals; (i) Methods for preparing hybridomas; (j) Any one of the methods described above; (k) any one of the methods (a) to (j) that is in vivo, in vitro, or ex vivo. An antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit for use in one or more of the above items. 116. The following: (a) Treatment, improvement, prevention, and / or diagnosis of cancer ((For example, as in Example 2 or Example 3 of this specification), preferably the cancer is selected from the group consisting of breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, cervical cancer, rectal cancer, colon cancer, esophageal cancer, stomach cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcoma, brain cancer, nevus and melanoma, genitourinary cancer, prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancer, retinoblastoma, thyroid cancer, fallopian tube cancer; more preferably the cancer is a solid tumor; most preferably the cancer is breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, uterine cancer (e.g., muscular cancer), cervical cancer, rectal cancer, colon cancer, anal cancer, esophageal cancer, Selected from the group consisting of stomach cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcoma (e.g., osteosarcoma and Kaposi's sarcoma), brain cancer (e.g., pituitary tumor), nevus and melanoma cancer, skin cancer (e.g., squamous cell carcinoma and melanoma), genitourinary cancer (e.g., ureteral and bladder cancer, testicular cancer, prostate cancer, penile cancer), prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancer (e.g., lymphoma, leukemia, myeloma, myelodysplastic syndrome, or myelofibrosis), eye cancer (e.g., retinoblastoma), neuroendocrine tumors, cancer of unknown primary origin (CUP), breast cancer, colon cancer, endometrial cancer, sarcoma, stomach cancer, head and neck cancer, pancreatic cancer, lung cancer, mesothelioma, and ovarian cancer); (b) Monitoring the progression of cancer, and / or evaluating the efficacy and / or toxicity of cancer treatments or anticancer compounds / agents; (c) Screening candidate compounds for their anticancer activity; (d) Altering the resistance of cancer cells to chemotherapy; (e) Making cancer cells sensitive to chemotherapy; (f) Inhibiting the proliferation of cancer cells that express TPBG; (g) Production or preparation of antibodies; (h) Immunizing non-human animals; (i) Preparation of hybridomas; (j) in any one of the above items; (k) In vivo, in vitro, or ex vivo use, any one of (a)-(j) Use of any one of the aforementioned antibodies, antibody-drug conjugates (ADCs), nucleic acids, expression vectors, host cells, compositions, or kits for one or more of the above. 117. An antibody-drug conjugate (ADC), composition, kit, method, and / or use of any of the aforementioned items, comprising or consisting of a dose (e.g., therapeutic dose, e.g., effective therapeutic dose, and / or daily dose, e.g., once daily dose or multiple daily doses, and / or total dose) of at least about 10 mg per kg of body weight of the subject to treatment (e.g., an animal or human patient), preferably about 10 to about 15 mg / kg (e.g., 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, or more) of any of the aforementioned items, or at least about 700 to 2000 mg in total of any of the aforementioned items (e.g., as in Example 2 or Example 3 of this Spec), 118. Any antibody-drug conjugate (ADC), composition, kit, method, and / or use of any of the above items, in which a dose of any one of the above items (e.g., therapeutic dose, e.g. effective therapeutic dose, and / or daily dose, e.g., once daily or multiple daily doses, and / or total dose) is administered (e.g., orally or intravenously) once or more times (e.g., two, three, four, five, six, seven, eight, nine, or ten times, e.g., every two or three days or once or twice a week) over a period of at least about one week (e.g., one to four weeks, e.g., three weeks, one month or two months), with or without dose escalation (e.g., as in Example 2 or Example 3 of this Spec). 119. An antibody-drug conjugate (ADC), composition, kit, method, and / or use of any of the above items, comprising or comprising one or more doses (e.g., two, three, four, five, six, seven, eight, nine, or ten times, e.g., every two or three days or once or twice a week) (e.g., orally or intravenously) of any one of the above items, administered (e.g., orally or intravenously) over a period of at least about one week (e.g., one to four weeks, e.g., three weeks, one month or two months), with or without dose escalation (e.g., as in Example 2 or Example 3 of this Spec). 120. An antibody-drug conjugate (ADC), composition, kit, method, and / or use of any of the above items, further comprising one or more therapeutic substances (e.g., anticancer agents or anti-cancer drugs). 121. An antibody-drug conjugate (ADC), composition, kit, method, and / or use of any one of the above items, further comprising the administration (pre-, concurrently, or post-) of one or more therapeutic substances (e.g., anticancer agents or anti-cancer drugs).

[0284] Where used herein, the singular forms “a,” “an,” and “the” refer to multiple objects unless otherwise indicated in the context. For example, “a reagent” refers to one or more of such reagents, and “the method” refers to equivalent steps and methods known to those skilled in the art, which may be modified or used in place of the method described herein.

[0285] Unless otherwise specified, the term “at least” preceding a series of elements should be understood to refer to all elements in that series. Those skilled in the art will recognize, or can verify through experiments not exceeding the scope of routine experimentation, numerous equivalents of the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0286] Whenever the terms “and / or” are used herein, they include the meanings of “and,” “or,” and “all or any other combination of the elements linked by the terms.”

[0287] When used herein, the terms "about" or "approximately" mean within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0288] The terms "less than" or "more than" do not include that specific number.

[0289] For example, "less than 20" means less than the specified number. Similarly, "more than" or "greater than" means more than or greater than the specified number; for example, "more than 80%" means more than or greater than the specified number, which is 80%.

[0290] Throughout this specification and the subsequent claims, unless otherwise indicated in the context, the word “comprise,” and variations such as “comprises” and “comprising,” shall be understood to mean encompassing the integer or process, or group of integers or processes, described herein, but not excluding any other integers and processes, or groups of integers and processes. Where used herein, the term “comprising” may be replaced by the terms “containing” or “including,” or sometimes, where used herein, the term “having.” Where used herein, “consisting of” excludes any element, process, or component not specified.

[0291] The term "includes" means "includes, but is not limited to." "Includes" and "includes, but is not limited to" are used synonymously.

[0292] It should be understood that the present invention is not limited to, and therefore may vary, the specific methodologies, protocols, materials, reagents, and substances described herein. The technical terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the present invention as defined solely by the claims.

[0293] All publications cited throughout this Specified, including all patents, patent applications, scientific publications, and instructions, whether preceding or following, are incorporated herein by reference in their entirety. Nothing herein should be construed as admitting that the present invention has no prior rights to such disclosures based on prior art. In the event that any material incorporated by reference conflicts with or is inconsistent with this Specified, this Specified shall prevail over any such material.

[0294] The contents of all documents and patent documents cited herein are incorporated in their entirety by reference. [Examples]

[0295] Examples of the Invention A deeper understanding of the present invention and its advantages will be evident from the following examples, which are provided for illustrative purposes only. These examples do not limit the scope of the present invention in any way.

[0296] Example 1: General information Chemicals, solvents, and antibodies Chemicals and solvents were purchased from Merck (Merck group, Germany), TCI (Tokyo Chemical Industry Co., Ltd., Japan), Iris Biotech (Iris Biotech GmbH, Germany), MCE (MedChemExpress, USA), and Carl Roth (Carl Roth GmbH + Co. KG, Germany), and used without further purification. Dry solvents were purchased from Merck (Merck group, Germany). PEG24 was purchased from BiochemPEG (Pure Chemistry Scientific Inc., United States).

[0297] Preparative HPLC For small-scale preparative HPLC, a VP 250 / 10 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany) was used in a BUCHI Pure C-850 Flash-Prep system (BUCHI Labortechnik AG, Switzerland). The following gradient was used: Method C: (A=H2O+0.1%TFA (trifluoroacetic acid), B=MeCN (acetonitrile)+0.1%TFA, flow rate 6 ml / min, 30%B 0-5 min, 30-70%B 5-35 min, 99%B 35-45 min). For larger scales, a VP 250 / 21 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany) was used, with the following gradient: Method D: (A=H2O+0.1%TFA (trifluoroacetic acid), B=MeCN (acetonitrile)+0.1%TFA, flow rate 14 ml / min, 30%B 0-5 min, 30-70%B 5-35 min, 99%B 35-45 min).

[0298] LC / MS Low molecular weight molecules, linker payloads, antibodies, and ADCs were analyzed using Waters H-class instruments equipped with a 4-solvent control system, Waters sample control system-FTN, Waters PDA detector, and Waters column control system, using an Acquity UPLC protein BEH C4 column (300 Å, 1.7 μm, 2.1 mm × 50 mm) for antibodies and ADCs. In this case, samples were eluted at a column temperature of 80°C. The following gradients were used: A: 0.1% formic acid dissolved in H2O, B: 0.1% formic acid dissolved in MeCN. Mass spectrometry was performed using a Waters XEVO G2-XS QTof analyzer. Proteins were ionized in positive ion mode by applying a 40kV cone voltage. Raw data were analyzed using MaxEnt 1. Small molecules and linker payloads were analyzed using an Acquity UPLC-BEH C18 column (300Å, 1.7μm, 2.1mm×50mm). In this case, the sample was eluted at a column temperature of 45°C and a flow rate of 0.4 mL / min. The following gradient was used: A: 0.1% formic acid dissolved in H2O, B: 0.1% formic acid dissolved in MeCN. 2%B 0-1 min, 2-98%B 1-5 min, 98%B 5-5.5 min, 98-2%B 5.5-6 min, 2%B 6-7 min.

[0299] Antibody synthesis and expression DNA encoding the light and heavy chains of the anti-TPBG antibody was synthesized (Geneart, Thermo Fisher), and the constant region of the heavy chain contained silencing mutations L234A and L235A (LALA mutations, see Figure 21). The signal sequences of the heavy chain (SEQ ID NO: 13, MDWTWRILFLVAAATGAHS) and the light chain (SEQ ID NO: 14, MLPSQLIGFLLLWVPASRG) were added. The light and heavy chain sequences were cloned into pcDNA3.4-TOPO (Thermo Fisher) expression plasmids. Next, the antibody was transiently expressed in Expi-CHO-S cells (Thermo Fisher) by co-transfecting cells with pcDNA3.4 expression plasmids (Thermo Fisher) encoding the heavy and light chains of each sequence in a 1:1 ratio using the Expi-CHO transfection system (Thermo Fisher). Cells were collected by centrifugation at 300g at 4°C for 5 minutes. To remove fine particles from the supernatant, the supernatant was centrifuged at 4000-5000g at 4°C for 30 minutes. For further clarification, the supernatant was passed through a 0.22 μm filter. Antibodies were purified from the clarified and filtered supernatant by protein A chromatography and analyzed by HPLC-SEC, HPLC-HIC, LC-MS, and SDS-PAGE.

[0300] Preparative size exclusion chromatography Protein purification by size exclusion chromatography was performed using an AKTA Pure FPLC system (GE Healthcare, United States) equipped with an F9-C fraction collector.

[0301] Measurement of ADC concentration ADC concentrations were measured in 96-well plates using the Pierce® Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific, USA) and Bradford Reagent B6916 (Merck, Germany) along with a pre-diluted bovine gamma globulin (Thermo Fisher Scientific, USA) protein assay standard. The results from both assay methods were arithmetically averaged.

[0302] Sample preparation of ADCs and antibodies for MS 0.5 μl of PNGase-F solution (Pomega, Germany, recombinant, cloned from Elizabethkingia miricola, 10 u / μl) and 5 μl of 100 mM DTT aqueous solution were added to 50 μl of PBS containing 0.2 mg / mL antibody or ADC, and the solution was incubated at 37°C for at least 2 hours. 2 μl of the solution was injected for each sample, and the samples were subjected to LC / MS.

[0303] Analytical size exclusion chromatography ADC analysis size exclusion chromatography (A-SEC) was performed on a Vanquish Flex UHPLC system equipped with a DAD detector, split sampler FT (4°C), column compartment H (25°C), and binary pump F (Thermo Fisher Scientific, USA), using a MAbPac SEC-1 300 Å, 4 × 300 mm column (Thermo Fisher Scientific, USA) at a flow rate of 0.15 mL / min. Separation of various ADC / mAb populations was achieved by a constant composition gradient over 30 minutes using pH 7 phosphate buffer (20 mM Na₂HPO₄ / NaH₂PO₄, 300 mM NaCl, 5% v / v isopropyl alcohol) as the mobile phase. 8 μg of ADC / mAb was loaded onto the column for A-SEC analysis. UV chromatograms were recorded at 220 nm and 280 nm.

[0304] Analytical Hydrophobic Interactions Chromatography Measurements were performed on a Vanquish Flex UHPLC system (2.9) using a MabPac HIC butyl 4.6 × 100 mm column (Thermo Fischer Scientific, USA). Separation of various ADCs / antibodies was achieved using the following gradient: A: 1M (NH4)2SO4, 500 mM NaCl, 100 mM NaH2PO4 pH 7.4, B: 20 ​​mM NaH2PO4, 20% (v / v) isopropyl alcohol, pH 7.4. 0%B: 0-1 min, 0-95%B: 1-15 min, 95%B: 15-20 min, 95-0%B: 20-23 min, 0%B: 23-25 ​​min, flow rate 700 μL / min. For each analysis, 15 μg of sample was loaded onto the column. UV chromatograms were recorded at 220 nm and 280 nm.

[0305] SDS-PAGE Samples were prepared for SDS-PAGE by incubation (95°C, 5 minutes) in SDS sample buffer (BioRad) supplemented with 25 mM DTT, separated using a 4-20% polyacrylamide gel (BioRad 4-20% Mini Protean TGX), and subsequently stained with Coomassi stain (Thermo Fisher Scientific Imperial protein stain).

[0306] ADC Synthesis: A common method for conjugating P5-based linker-payload constructs to obtain DAR8. 50 μl of anti-TPBG antibody (containing Fc silencing mutations such as leucine (L) to alanine (A) substitution at positions 234 and 235 (LALA mutation)) in a 10.0 mg / ml solution dissolved in P5-conjugation buffer (50 mM Tris, 1 mM EDTA, 100 mM NaCl, pH 8.3, room temperature) was mixed with 3.33 μl of 10 mM TCEP solution in P5-conjugation buffer. Immediately afterward, 1.67 μl of a 40 mM solution of P5-exatecan construct dissolved in DMSO was added. This mixture was shaken at 350 rpm and 25°C for 16 hours. The reaction mixture was purified by preparative size exclusion chromatography using 25 ml of Superdex® 200 Increase 10 / 300GL (Cytiva, Sweden) and eluted with sterile PBS (Merck, Germany) at a flow rate of 0.8 ml / min. The antibody-containing fraction was combined and concentrated by spin filtration (Amicon® Ultra-2 mL MWCO: 30 kDa, Merck, Germany).

[0307] Binding to human 5T4 was evaluated by flow cytometry. Equilibrium binding constant (K) for endogenously expressed human 5T4 D To determine the 5T4-expressing MDA-MB-468 cells, they were incubated with antibodies at concentrations ranging from 0.002 to 200 nM, stained with Alexa-labeled anti-human IgG H+L secondary antibody (Thermo Fisher Scientific), and analyzed by flow cytometry. The mean fluorescence intensity (MFI) ratio was normalized relative to a nonspecific binding control. The assay was performed in pairs, and data points were analyzed by nonlinear regression using a single-site specific binding model, using Prism 9 software. D The value was obtained. In MDA-MB-468 cells, the K of the anti-TBPG antibody D The value was measured at 0.2473 nM. The graph shows the mean (n=2) ± SEM (Figure 1).

[0308] Binding to human 5T4, as evaluated by ELISA. The binding of gradually increasing concentrations of anti-TPBG antibody to purified, immobilized recombinant human 5T4 antigen was investigated using an ELISA setting.

[0309] To measure the binding of anti-TPBG antibodies to the 5T4 antigen, 96-well, F-bottom, black MaxiSorp, Nunc-Immuno plates (Thermo Fisher) were coated with 1 μg / mL of purified recombinant human 5T4 antigen, consisting of the extracellular domain (ACD) of 5T4 expressed as a 6× histidine fusion protein in Expi-HEK293 cells. For deglycosylation, the antigen was incubated overnight at 37°C with PNGase F (Promega). Deglycosylation was confirmed by Coomassi-stained SDS-PAGE (Figure 2B). After blocking with 2% bovine serum albumin (Carl Roth) in 1×PBS-Tween20 (0.05%), the antibody concentration was increased (0.00015 μg / ml, 0.00046 μg / ml, 0.00137 μg / ml, 0.00412 μg / ml, 0.01235 μg / ml, 0.03704 μg / ml, 0.11111 μg / ml, 0.33333 μg / ml, 1.00000 μg / ml, 3.00000 μg / ml, 9.00000 μg / ml) and conjugated at room temperature for 2 hours. The bound antibody was detected by incubation at room temperature for 1 hour with alkaline phosphatase AffiniPure rabbit anti-human IgG (Fcγ fragment specific) (Jackson Immunoresearch, 309-055-008) (diluted 1:5000 with blocking solution). A 4-methylumbelliferyl phosphate (4-MUP) (Merck) substrate was added, and after incubation at room temperature for 15 minutes, 25 μl / well of 3M NaOH was added. Hydrolysis of 4-MUP to the soluble fluorescent substance methylumbelliferone was quantified based on the fluorescence signal of methylumbelliferone (excitation wavelength 385 nm, emission wavelength 448 nm) using a microplate reader Infinite M1000 Pro (Tecan). The apparent dissociation constant (K) was then determined. D ) was calculated using nonlinear regression with a single-site specific binding model with Prism 9 software. DThe values ​​were 0.10 nM for glycosylated 5T4 and 0.16 nM for deglycosylated 5T4. The graph shows the mean (n=2) ± SEM (Figure 2A).

[0310] The binding to 5T4 in marmosets with white tufts around the ears was evaluated by flow cytometry. HEK293 cells were transiently transfected with expression plasmids encoding full-length 5T4-(GGGGS)-mCherry from humans, cynomolgus monkeys, or marmosets. Equilibrium binding constant (K) D To determine the optimal antibody-positive cell type (mCherry), HEK293 cells transiently expressing full-length 5T4-mCherry from humans, cynomolgus monkeys, or marmosets were incubated with antibodies ranging from 0.002 to 200 nM. These cells were stained with Alexa-labeled anti-human IgG H+L secondary antibody (Thermo Fisher Scientific) and analyzed by flow cytometry. Antibody binding was investigated in mCherry-positive cells. The mean fluorescence intensity (MFI) ratio was normalized relative to the secondary antibody control. The assay was performed in pairs, and data points were analyzed by nonlinear regression using a single-site specific binding model, using Prism 9 software. D The value was obtained. K D The nM (nM) was 2.155 for humans, not applicable for cynomolgus monkeys, and 5.455 for marmosets. The graph shows the mean (n=2) ± SD (Figure 3).

[0311] Internal migration evaluated by flow cytometry To investigate internal migration using pHrodo, antibodies were labeled using the pHrodo® Deep Red Antibody Labeling Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. 5T4-positive (BXPC-3) and 5T4-negative (SW-620) cells were incubated with 5 μg / ml of pHrodo-labeled antibody at 37°C for 1 hour, 5 hours, and 24 hours. An increase in MFI indicates the presence of antibodies in the late endosomal and lysosomal compartments. The MFI ratio was determined by dividing the MFI of cells incubated with pHrodo by the MFI of unstained cells (Figure 4).

[0312] In vitro cytotoxicity assessed by the resazurin assay. To investigate the direct cytotoxicity of ADC, each cell was seeded in a 96-well plate (flat-bottom, 5000 cells / well, suspended in 100 μl of culture medium) and incubated with increasing concentrations of ADC in the medium (0-3 μg / ml) for 7 days to create a dose-response curve. Before viability analysis, the supernatant on adherent cells was removed and replaced with fresh medium. Subsequently, cell death was analyzed using resazurin (Sigma-Aldrich) at a final concentration of 55 μM as a cell viability discriminant dye. Fluorescence emission at 590 nM was measured using a microplate reader Infinite M1000 Pro (Tecan). Cell viability was determined by dividing the fluorescence of ADC-treated cells by the fluorescence of control cells similarly treated with culture medium alone. Data points were analyzed by nonlinear regression using a single-site-directed binding model, and IC was performed using Prism9 software. 50 The value was obtained. TUB-030 ADC IC 50 The (ng / mL) values ​​were 34.3 ng / mL (MDA-MB-468), 89.0 ng / mL (BXPC-3), and 72.3 ng / mL (DU-145). Minimum survival rate (E Max The percentages were 10.41% (MDA-MB-468), 16.8% (BXPC-3), and 15.3% (DU-145). The graph shows the mean (n=2) ± SEM (Figure 5).

[0313] ADC bystander activation To analyze the bystander activity of ADC against target-negative cells, 20,000 5T4-positive cells (BXPC-3) were incubated with gradually increasing concentrations of ADC (0-3 μg / ml). After 5 days, half of the cell culture supernatant was transferred to 5,000 5T4-negative cells (SW-620) and incubated for another 5 days. Cell death was analyzed by measuring viability using resazurin as described above (Figure 6).

[0314] In vitro inhibition of topoisomerase I by delivery of exatecan via ADC. Inhibition of topoisomerase I by exatecan delivery via TUB-030 ADC induces DNA damage markers. MDA-MB-468 cells were treated with 5 μg / mL TUB-030 or 5 nM free exatecan for 24, 48, and 72 hours. Cells were stained for the DNA damage markers active caspase-3, cleaved PARP, and phosphorylated H2A.X (Ser-139) and analyzed by flow cytometry. Graphs show mean (n=2) ± SEM (Figure 7).

[0315] In vivo efficacy of ADC in mouse xenograft models All animal experiments were conducted in accordance with German animal welfare laws and approved by local authorities. In short, 1 x 10 7 Individual MDA-MB-468 cells were subcutaneously injected into CB17-Scid mice. The tumor size was 0.188 cm² on day 28 after transplantation. 3 Treatment was initiated when the mean tumor volume reached [a certain level]. After randomly dividing animals into treatment and control groups, five animals in each group were treated once intravenously with either anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR 8 or a vehicle at a dose of 1 mg / kg, 3 mg / kg, or 10 mg / kg. Tumor volume, body weight, and overall health status were recorded throughout the study (Figures 8-9).

[0316] In vivo PK evaluation of ADC In vivo PK experiments were conducted using anti-TPBG-P5(PEG24)-VC-PAB-exatecan. Female Sprague Dolly rats were treated with 10 mg / kg of unconjugated TPBG antibody or ADC. Blood samples were collected at various time points, and the amount of ADC was quantified using total antibody ELISA and intact ADC ELISA assays. To evaluate the in vivo PK of ADC, the total antibody concentration in the serum of ADC-treated SD rats was measured at various time points. All humanized anti-5T4 antibodies were analyzed in rat serum in the range of 2000–15.6 ng / ml. Nunc 96-well plates (100 μl / well) were coated with PBS-diluted 5T4 (required concentration: 0.25 μg / ml) and sealed with PCR foil. The plates were incubated in a refrigerator and maintained at a temperature of 2–8°C overnight. The coated plates were washed three times with 300 μl of PBST. 200 μl / well of blocking solution (2% albumin mixed in PBST) was added, the plate was sealed, and incubated at room temperature for 1 hour. The coated plate was washed three times with 300 μl of PBST. 100 μl of the prepared standards (2000-15.6 ng / ml each of ADC, QC, and test samples) was added per well, the plate was sealed, and incubated at room temperature for 1 hour. The plate was washed three times with 300 μl of PBST. 100 μl / well of anti-human IgG (γ-chain specific)-peroxidase antibody (diluted 1:60000 in PBS) was added and incubated at room temperature for 1 hour. The plate was washed three times with 300 μl of PBST. 50 μl / well of TMB was added, the plate was sealed, and incubated at room temperature for 15 minutes. 50 μl / well of 1 M sulfuric acid was added. Absorbance at a wavelength of 450 nm was measured using a Tecan plate reader.

[0317] To evaluate the in vivo stability of ADC, the concentration of intact ADC in the serum of ADC-treated SD rats was measured at various time points. Intact ADC was analyzed in rat serum in the range of 2000–15.6 ng / ml. Nunc 96-well plates (100 μl / well) were coated with rabbit anti-exatecan mAb (required concentration: 1 μg / ml) diluted in PBS and sealed with PCR foil. The plates were incubated in a refrigerator, maintaining a temperature of 2–8°C overnight. The coated plates were washed three times with 300 μl of PBST. 200 μl / well of blocking solution (2% albumin mixed with PBST) was added, the plates were sealed, and incubated at room temperature for 1 hour. The coated plates were washed three times with 300 μl of PBST. Prepared standards (2000–15.6 ng / ml each for ADC, QC, and test samples) were added at a rate of 100 μl per well, the plate was sealed, and incubated at room temperature for 1 hour. The plate was washed three times with 300 μl of PBST. 100 μl / well of goat anti-human IgG (H+L) (adsorbed) (diluted 1:25000 in PBS) was added and incubated at room temperature for 1 hour. The plate was washed three times with 300 μl of PBST. 100 μl / well of TMB was added, the plate was sealed, and incubated at room temperature for 10 minutes. 100 μl / well of 1 M sulfuric acid was added. Absorbance at a wavelength of 450 nm was measured using a Tecan plate reader (Figure 10).

[0318] Linker-payload synthesis General method 2 for synthesizing PEGylated P5 structural units by Staudinger phosphonite reaction TIFF2026513098000229.tif56148

[0319] In a 25 ml Schlenk flask, under an argon atmosphere, 267 mg of bis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 equivalent) was added, cooled to 0°C, and 2.20 mL of ethinyl magnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 equivalent) was added dropwise. The yellowish solution was raised to room temperature and stirred for a further 30 minutes. 3.00 mmol (3.0 equivalent) of the desired PEG alcohol, dissolved in 5.56 mL of 1H tetrazole solution (0.45 M in MeCN, 2.50 mmol, 2.50 equivalent), was added, and the white suspension was stirred overnight at room temperature. To form the desired phosphonite, 31 The reaction was monitored by P-NMR. 1.0 mmol (1.0 equivalent) of the desired azide dissolved in 2 mL of DMF, THF, or MeCN was added, and the suspension was stirred at room temperature for 24 hours. The crude reaction mixture was purified by preparative HPLC.

[0320] P5(PEG12)-OSu TIFF2026513098000230.tif26128

[0321] The title compound was synthesized according to General Method 2 from 19.5 mg of bis(diisopropylamino)chlorophosphine (73 μmol, 1.00 equivalent), 146 μL of ethynylmagnesium bromide solution (0.5 M, 73 μmol, 1.00 equivalent in THF), 100 mg of dodecaethylene glycol (183 μmol, 2.50 equivalent), 400 μL of 1H-tetrazole solution (0.45 M, 183 μmol in MeCN), and 19 mg of 4-azidobenzoic acid-N-hydroxysuccinimide (73 μmol, 1.00 equivalent). After preparative HPLC (Method D) and lyophilization, the product was obtained as a colorless oil (42.5 mg, 50 μmol, 68%). TIFF2026513098000231.tif45165

[0322] P5(PEG12)-COOH TIFF2026513098000232.tif24128

[0323] The title compound was synthesized according to General Method 2 from 40 mg of bis(diisopropylamino)chlorophosphine (150 μmol, 1.00 equivalent), 360 μL of ethinyl magnesium bromide solution (0.5 M, 180 μmol, 1.2 equivalents in THF), 245 mg of PEG12 (450 μmol, 3.0 equivalents), 0.83 mL of 1H-tetrazole solution (0.45 M, 450 μmol, 2.5 equivalents in MeCN), and 39 mg of 4-azidobenzoic acid (150 μmol, 1.00 equivalent). After preparative HPLC (Method D) and lyophilization, the product was obtained as a colorless oil (25 mg, 34 μmol, 23%). HR-MS result: C 33 H 57 NO 16 P + [M+H] + Theoretical value: 754.3410, measured value: 754.3398 (Figure 11).

[0324] P5(PEG24)-OSu TIFF2026513098000233.tif27128

[0325] The title compound was synthesized according to General Method 2 from 41 mg of bis(diisopropylamino)chlorophosphine (159 μmol, 1.00 equivalent), 370 μL of ethinyl magnesium bromide solution (0.5 M, 185 μmol, 1.2 equivalents in THF), 450 mg of PEG24 (388 μmol, 2.50 equivalents), 1.02 mL of 1H-tetrazole solution (0.45 M, 466 μmol, 3.0 equivalents in MeCN), and 40 mg of 4-azidobenzoic acid N-hydroxysuccinimide (155 μmol, 1.00 equivalent). After preparative HPLC (Method D) and lyophilization, the product was obtained as a colorless oil (79 mg, 57 μmol, 37%). MS results: C 61 H 109 N2O 30 P 2+ [M+2H] 2+ Theoretical value: 690.3396, measured value: 690.81. (Figure 12).

[0326] NH2-VC-PAB-exatecan TFA salt TIFF2026513098000234.tif51152

[0327] 34.3 mg of exatecan mesylate (0.0645 mmol, 1.0 equivalent) was placed in a screw-cap vial and suspended in 645 μL of dry DMSO. 241 μL (0.0967 mmol, 1.5 equivalents) of 0.4 mol / L Fmoc-VC-PAB-PNP solution in dry DMSO, 64.5 μL (0.0645 mmol, 1.0 equivalent) of 1 mol / L HOBt hydrate solution in dry DMSO, and 113 μL (0.645 mmol, 10.0 equivalents) of DIPEA were added. The yellow solution was stirred at 50°C for 2 hours. Then, 425 μL of 50% (w / w) diethanolamine solution in dry DMSO was added, and the reaction mixture was stirred at room temperature for a further 30 minutes. 1.5 ml of MeCN and 2.5 mL of H2O were added, and the resulting yellow solution was directly purified by preparative HPLC using Method D. After lyophilization, 47.3 mg (76.7%, 0.0495 mmol) of a yellowish solid was obtained as TFA salt. HR-MS result: C 43 H 50 FN8O9 + [M+H] + Theoretical value: 841.3680, measured value: 841.3696 (Figure 13).

[0328] NH2-VA-PAB-exatecan TFA salt TIFF2026513098000235.tif53157

[0329] 1.23 mg of exatecan mesylate (0.00232 mmol, 1.0 equivalent) was placed in a screw-cap vial and suspended in 23 μL of dry DMSO. 8.7 μL (0.00348 mmol, 1.5 equivalents) of 0.4 mol / L Fmoc-VA-PAB-PNP solution in dry DMSO, 2.3 μL (0.00232 mmol, 1.0 equivalent) of 1 mol / L HOBt hydrate solution in dry DMSO, and 4 μL (0.0232 mmol, 10.0 equivalents) of DIPEA were added. The yellow solution was stirred overnight at room temperature. Then, 15 μL of 50% (w / w) diethanolamine solution in dry DMSO was added, and the reaction mixture was stirred for a further 30 minutes at room temperature. 1.5 ml of MeCN and 2.5 mL of H2O were added, and the yellow solution was directly purified by preparative HPLC using Method C. After freeze-drying, a yellowish solid was obtained containing 1.01 mg (50.0%, 0.00116 mmol) of TFA salt. HR-MS result: C 40 H 44 FN6O8 + [M+H] + Theoretical value: 755.3200, measured value: 755.3201 (Figure 14).

[0330] P5(PEG2)-VC-PAB-Exatecan TIFF2026513098000236.tif54158

[0331] 23.4 μL (0.00468 mmol, 1.0 equivalent) of 200 mM NH2-VC-PAB-exatecan TFA salt solution in dry DMSO, 46.8 μL (P5(PEG2)-COOSu, 0.00936 mmol, 2.0 equivalent) of 200 mM 2-(2-hydroxyethoxy)ethyl-N-(4-benzoic acid-N-hydroxysuccinimide)-P-ethynylphosphoneamidate solution, and 4.08 μL (0.0234 mmol, 5.0 equivalent) of DIPEA were placed in a screw-cap vial. The solution was shaken at 50°C for 5 hours, allowed to cool to room temperature, and 1.5 ml of MeCN and 2.5 mL of H2O were added. This solution was directly purified by preparative HPLC using Method C. After freeze-drying, 1.33 mg (25.0%, 0.00117 mmol) of a yellowish solid was obtained. HR-MS result: C 56 H 64 FN9O 14 P + [M+H] + Theoretical value: 1136.4289, measured value: 1136.4306. (Figure 15).

[0332] P5(PEG12)-VC-PAB-Exatecan TIFF2026513098000237.tif54158

[0333] 51 μL (0.0102 mmol, 1.0 equivalent) of 200 mM NH2-VC-PAB-exatecan TFA salt solution in dry DMSO, 102 μL (P5(PEG12)-COOH, 0.0204 mmol, 2.0 equivalent) of 200 mM PEG12-N-(4-benzoic acid)-P-ethynylphosphoneamidate solution in dry DMSO, 102 μL (0.0255 mmol, 2.5 equivalent) of 250 mM Pybop solution in dry DMSO, and 8.89 μL (0.051 mmol, 5.0 equivalent) of DIPEA were placed in a screw-cap vial. The solution was shaken at room temperature for 2 hours, and 1.5 ml of MeCN and 2.5 mL of H2O were added. This solution was directly purified by preparative HPLC using Method D. After freeze-drying, 15.91 mg (99.0%, 0.0101 mmol) of a yellowish solid was obtained. HR-MS result: C 76 H 105 FN9O 24 P 2+ [M+H] 2+ Theoretical value: 788.8492, measured value: 788.8485. (Figure 16).

[0334] P5(PEG24)-VC-PAB-Exatecan TIFF2026513098000238.tif54156

[0335] 102 μL (0.0204 mmol, 1.0 equivalent) of a 200 mM solution of NH2-VC-PAB-exatecan TFA salt in dry DMSO, 204 μL (0.0408 mmol, 2.0 equivalents) of a 200 mM solution of P5(PEG24)-OSu in dry DMSO, and 17.78 μL (0.102 mmol, 5.0 equivalents) of DIPEA were placed in a screw-cap vial. The solution was shaken overnight at room temperature, and 1.5 ml of MeCN and 2.5 mL of H2O were added. This solution was directly purified by preparative HPLC using Method D. After lyophilization, 25.76 mg (60.0%, 0.01224 mmol) of a yellowish solid was obtained. HR-MS result: C 100 H 153 FN9O 36 P 2+ [M+H] 2+ Theoretical value: 1053.5081, measured value: 1053.50833. (Figure 17).

[0336] P5(PEG12)-VA-PAB-Exatecan TIFF2026513098000239.tif53155

[0337] In a screw-cap vial, 11.6 μL (0.00116 mmol, 1.0 equivalent) of 100 mM NH2-VA-PAB-exatecan TFA salt solution in dry DMSO, 8.7 μL (P5(PEG12)-COOH, 0.00174 mmol, 1.5 equivalent) of 200 mM PEG12-N-(4-benzoic acid)-P-ethynylphosphoneamidate solution in dry DMSO, 11.6 μL (0.00232 mmol, 2.0 equivalent) of 200 mM Pybop solution in dry DMSO, and 2.02 μL (0.0116 mmol, 10.0 equivalent) of DIPEA were added. The solution was shaken at room temperature for 2 hours, and 1.5 ml of MeCN and 2.5 mL of H2O were added. This solution was directly purified by preparative HPLC using Method C. After freeze-drying, 0.56 mg (32.2%, 0.000375 mmol) of a yellowish solid was obtained. HR-MS result: C 73 H 99 FN7O 23 P 2+ [M+H] 2+ Theoretical value: 745.8252, measured value: 745.8255. (Figure 18).

[0338] P5 (PEG12)-exatecan TIFF2026513098000240.tif56140

[0339] 50 μL (0.005 mmol, 1.0 equivalent) of a 100 mM exatecan mesylate suspension in dry DMSO, 20 μL (P5(PEG12)-COOH, 0.005 mmol, 1.0 equivalent) of a 250 mM PEG12-N-(4-benzoic acid)-P-ethynylphosphoneamidate solution in dry DMSO, 20 μL (0.006 mmol, 1.2 equivalents) of a 300 mM Pybop solution in dry DMSO, and 4.33 μL (0.025 mmol, 5.0 equivalents) of DIPEA were placed in a screw-cap vial. The solution was shaken at room temperature for 2 hours, and 1.5 ml of MeCN and 2.5 mL of H2O were added. This solution was directly purified by preparative HPLC using Method C. After freeze-drying, 2.63 mg (45.0%, 0.0023 mmol) of a yellowish solid was obtained. HR-MS result: C 57 H 77 FN4O 19 P + [M+H] + Theoretical value: 1171.4899, measured value: 1171.4852. (Figure 19).

[0340] result Analysis of ADC synthesized from P5(PEG24)-VC-PAB-exatecan (Figure 20) Analytical characterization of ADC synthesized from anti-TPBG antibody conjugated to P5(PEG24)-VC-PAB-exatecan and purified by CEX. A) SEC for analysis, B) HIC for analysis, C) MS spectrum after conjugation. The data clearly show that the ADC was completely conjugated up to DAR8 and that only a small amount of aggregates were present after purification (Figure 20).

[0341] Example 2: Ex vivo serum stability, in vivo efficacy in patient-derived xenograft models (PDX), and in vivo toxicity of an exemplary ADC of the present invention: Ex vivo serum stability: Serum samples from each species were spiked with 0.2 mg / ml of anti-TPBG-P5(PEG24)-VC-PAB-exatecan, mixed with at least 80% serum. The samples were sterile filtered using a UFC30GV0S centrifugal filter unit (Merck, Germany) and incubated at 37°C for 1, 2, 3, 5, and 7 days. Samples on day 0 were processed further without modification.

[0342] Recombinant 5T4 antigen was conjugated to Thermo NHS magnetic beads according to the manufacturer's instructions. The bead storage solution was removed from 40 μl of 5T4-conjugated bead suspension. These beads were incubated with 100 μl of serum-ADC mixture pre-mixed with 200 μl of PBS for 2 hours at room temperature. The supernatant was then removed, and the resin was washed twice with 1 mL of PBS-T. Subsequently, the beads were incubated with 10 μl of 100 mM glycine buffer (pH 2.5) for 15 minutes at room temperature. This solution was rebuffered in PBS using a 75 μL Zeba® spin desalting column (Thermo Fisher Scientific, USA) at 7K MWCO. The samples were further processed for MS analysis as described above. The drug-to-antibody ratio (DAR) was calculated from the MS intensity of light chain adducts conjugated to 0 or 1 molecule of P5(PEG24)-VC-PAB-exatecan and the MS intensity of heavy chain adducts conjugated to 0 to 3 molecules of P5(PEG24)-VC-PAB-exatecan.

[0343] These results clearly demonstrate that the linker between the anti-TPBG antibody and the exatecan drug molecule is highly stable in the serum of various species, and that the amount of payload does not significantly decrease (no change in drug-to-antibody ratio (DAR)) even after incubation of the ADC for several days (Figure 22).

[0344] In vivo efficacy of ADCs in patient-derived xenograft models (PDX): All animal experiments were conducted in accordance with German animal welfare laws and approved by local authorities. In short, 3x3 mm samples of tumor tissue derived from patients were subcutaneously transplanted into the flanks of immunodeficient female NMRI nu / nu mice. The tumors were 0.1–0.15 cm in size. 3 Treatment was initiated when the mean tumor volume reached 10 mg / kg. Animals in each group were treated once on day 0 by intravenous injection with 10 mg / kg of anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR8, isotype-P5(PEG24)-VC-PAB-exatecan DAR8, or vehicle. Tumor volume, body weight, and overall health status were recorded throughout the study.

[0345] The data clearly demonstrate significantly higher in vivo efficacy in patient-derived tumor models compared to vehicle controls in various tumor types, including lung cancer, endometrial cancer, breast cancer, colon cancer, gastric cancer, head and neck cancer, pancreatic cancer, and ovarian cancer, as well as sarcomas and mesotheliomas. Highest selectivity was observed in models where isotype-controlled ADCs showed little to no antitumor activity at the same dose (Figure 23).

[0346] In vivo toxicity in marmosets: Toxicological studies were conducted in marmoset monkeys. At least two animals in each group were intravenously administered 10 mg / kg and 15 mg / kg of anti-TPBG-P5(PEG24)-VC-PAB-exatecan on days 1 and 22, and final dissection was performed on day 43. Total and intact ADCs in animal plasma samples were analyzed by ELISA. No significant changes in body weight, clinical chemistry, or hematological tests were observed at these dose levels (Figure 24).

[0347] The dose applied was about 10 times higher than the doses investigated in toxicological studies of other 5T4 targeting ADCs.

[0348] The significant improvement in ADC tolerability may be due to the reduced tendency of ADC aggregation as described herein. Antibody and ADC aggregation has been shown to be one of the major off-target toxicity promoters of ADC. Furthermore, Fc silencing introduced into the anti-TPBG antibody as described herein may also contribute to reduced toxicity in toxicological experiments and, consequently, increased tolerability. This important preclinical toxicity experiment to assess the safety of the molecule was made possible thanks to the aforementioned cross-reactivity of the anti-TPBG antibody against marmoset 5T4. Without cross-reactivity with marmosets, meaningful preclinical data could not have been obtained before initiating clinical trials in humans.

[0349] Example 3: Further Characterization of Anti-TPBG and ADCs Based Thereon For all data presented, anti-TPBG-P5(PEG24)-VC-PAB-exatecan refers to the LALA variant of the anti-TPBG mAb.

[0350] Synthesis and expression of comparative antibodies Anti-TPBG comparative antibody 1: Light chain of anti-TPBG comparative antibody 1 TIFF2026513098000241.tif31166 and heavy chain DNA encoding TIFF2026513098000242.tif58166 was synthesized (Geneart, Thermo Fisher). The heavy chain signal sequence (SEQ ID NO: 17, MDWTWRILFLVAAATGAHS) and the light chain signal sequence (SEQ ID NO: 18, MLPSQLIGFLLLWVPASRG) were added. The light and heavy chain sequences were cloned into pcDNA3.4-TOPO (Thermo Fisher) expression plasmids. Next, antibodies were transiently expressed in Expi-CHO-S cells (Thermo Fisher) by co-transfecting cells with pcDNA3.4 expression plasmids (Thermo Fisher) encoding the heavy and light chains of each sequence in a 1:1 ratio using the Expi-CHO transfection system (Thermo Fisher). Cells were collected by centrifugation at 300g, 4°C for 5 minutes. To remove particulate matter from the supernatant, the supernatant was centrifuged at 4000-5000 g at 4°C for 30 minutes. For further clarification, the supernatant was passed through a 0.22 μm filter. Antibodies were purified from the clarified and filtered supernatant by protein A chromatography.

[0351] Anti-TPBG comparative antibody 2: Light chain of anti-TPBG comparative antibody 2 TIFF2026513098000243.tif31170 and heavy chain DNA encoding TIFF2026513098000244.tif58166 was synthesized (Geneart, Thermo Fisher). The heavy chain signal sequence (SEQ ID NO: 21, MDWTWRILFLVAAATGAHS) and the light chain signal sequence (SEQ ID NO: 22, MLPSQLIGFLLLWVPASRG) were added. The light and heavy chain sequences were cloned into pcDNA3.4-TOPO (Thermo Fisher) expression plasmids. Next, antibodies were transiently expressed in Expi-CHO-S cells (Thermo Fisher) by co-transfecting cells with pcDNA3.4 expression plasmids (Thermo Fisher) encoding the heavy and light chains of each sequence in a 1:1 ratio using the Expi-CHO transfection system (Thermo Fisher). Cells were collected by centrifugation at 300g, 4°C for 5 minutes. To remove particulate matter from the supernatant, the supernatant was centrifuged at 4000-5000 g at 4°C for 30 minutes. For further clarification, the supernatant was passed through a 0.22 μm filter. Antibodies were purified from the clarified and filtered supernatant by protein A chromatography.

[0352] Synthesis of comparative ADCs Anti-TPBG comparison ADC1: 50 μl of 10 mg / mL anti-TPBG comparative antibody solution (66.67 μM) in Dulbecco's PBS (Merck KGaA) was mixed with 1.33 μl of TCEP solution (0.5 mM in buffer, Merck KGaA, diluted to 10 mM in PBS, 4 equivalents of TCEP relative to the antibody). After incubation at room temperature for 30 minutes, 0.83 μl of 40 mM maleimidocaproylmonomethylaulistatin F (MC-MMAF) solution in DMSO (10.0 equivalents relative to the antibody) was added. The mixture was shaken at room temperature (25°C) for 1 hour (350 RPM). The reaction mixture was purified by preparative size exclusion chromatography using 25 ml of Superdex® 200 Increase 10 / 300GL (Cytiva, Sweden) and eluted with sterile PBS (Merck, Germany) at a flow rate of 0.8 ml / min. As described in Example 1, the antibody-containing fractions were combined, concentrated by spin filtration (Amicon® Ultra-2mL MWCO:30kDa, Merck, Germany), and analyzed by MS.

[0353] Figure 25 shows the MS analysis of anti-TPBG comparative ADC1 synthesized by the method described above. The signal is annotated with the measured mass (in Daltons) and absolute intensity. The drug-to-antibody ratio was estimated to be 4.2 from the MS signal. LC: light chain of anti-TPBG comparative antibody 1, HC: heavy chain of anti-TPBG comparative antibody 1.

[0354] Anti-TPBG comparison ADC2: 50 μl of 10 mg / mL anti-TPBG comparative antibody solution 2 (66.67 μM) in Dulbecco's PBS (Merck KGaA) was mixed with 13.3 μl of TCEP solution (0.5 mM Merck KGaA in buffer, diluted to 10 mM in PBS, 40 equivalents of TCEP relative to the antibody). After incubation at room temperature for 1 hour, the solution was rebuffered in PBS using a 0.5 mL Zeba® spin desalting column (Thermo Fisher Scientific, USA) at 7K MWCO according to the manufacturer's instructions. 13.33 μl of 10 mM dehydroascorbic acid (Merck KGaA, 20 mM in PBS, 40 equivalents relative to the antibody) was added, and the mixture was incubated at room temperature for 3 hours. Subsequently, 6.66 μl of 10 mM MC-VC-SECO-DUBA solution dissolved in DMSO (MedChemExpress, 20 equivalents relative to the antibody) was added. The mixture was shaken at room temperature (25°C) for 1 hour (350 RPM). The reaction mixture was purified by preparative size exclusion chromatography using 25 ml of Superdex® 200 Increase 10 / 300GL (Cytiva, Sweden) and eluted with sterile PBS (Merck, Germany) at a flow rate of 0.8 ml / min. As described in Example 1, the antibody-containing fraction was combined, concentrated by spin filtration (Amicon® Ultra-2 mL MWCO: 30 kDa, Merck, Germany), and analyzed by MS.

[0355] Figure 26 shows the MS analysis of anti-TPBG comparative ADC2 synthesized by the method described above. The signal is annotated with the measured mass (in Daltons) and absolute intensity. The drug-to-antibody ratio was estimated to be 1.7 from the MS signal. LC: light chain of anti-TPBG comparative antibody 2, HC: heavy chain of anti-TPBG comparative antibody 2.

[0356] In vitro and in vivo experiments: In vitro cytotoxicity evaluated by the resazurin assay—comparative to anti-TPBG ADC1 and anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR8: To investigate the direct cytotoxicity of ADC, each cell was seeded in a 96-well plate (flat-bottom, 5000 cells / well, suspended in 100 μl of medium) and incubated with increasing concentrations of ADC in the medium (0–12 μg / ml) for 7 days to create a dose-response curve. Before viability analysis, the supernatant on adherent cells was removed and replaced with fresh medium. Subsequently, cell death was analyzed using resazurin (Sigma-Aldrich) at a final concentration of 55 μM as a cell viability discriminant dye. Fluorescence emission at 590 nM was measured using a microplate reader Infinite M200 Pro (Tecan). Cell viability was determined by dividing the fluorescence of ADC-treated cells by the fluorescence of control cells similarly treated with medium alone. The graph shows the mean (n=2) ± SEM.

[0357] In this experiment, the cytotoxicity of anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR 8, which targets 5T4, was investigated compared to anti-TPBG-comparative ADC1. In all tested cell lines with varying TPBG expression levels, anti-TPBG-P5(PEG24)-VC-PAB-exatecan showed an 18- to 66-fold improvement in cell death IC50 compared to anti-TPBG-comparative ADC1.

[0358] When comparing IC50 values ​​with anti-TPBG-comparative ADC1, the killing of antigen-positive tumor cells by anti-TPBG-P5(PEG24)-VC-PAB-exatecan was 18 to 66 times more effective. Specificity was demonstrated because isotype-controlled ADCs did not show efficacy. These results clearly indicate that the conjugates disclosed herein are superior to previously developed ADCs targeting the same target.

[0359] Figure 27 shows the cytotoxic dose-response of anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR 8 and its corresponding non-targeting isotype control conjugate compared to anti-TPBG comparison ADC1 in four different cell lines. The mean and standard deviation of the two measurements, as well as the dose-response fitting, are shown. Fluorescence (in %) compared to the medium control is equal to the percentage of viable cells (%).

[0360] In vitro cytotoxicity of ADC2 and anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR8 as evaluated by the Resazurin assay. To investigate the direct cytotoxicity of ADC, each cell was seeded in a 96-well plate (flat-bottom, 5000 cells / well, suspended in 100 μl of medium) and incubated with increasing concentrations of ADC in the medium (0–12 μg / ml) for 7 days to create a dose-response curve. Before viability analysis, the supernatant on adherent cells was removed and replaced with fresh medium. Subsequently, cell death was analyzed using resazurin (Sigma-Aldrich) at a final concentration of 55 μM as a cell viability discriminant dye. Fluorescence emission at 590 nM was measured using a microplate reader Infinite M200 Pro (Tecan). Cell viability was determined by dividing the fluorescence of ADC-treated cells by the fluorescence of control cells similarly treated with medium alone. The graph shows the mean (n=2) ± SEM.

[0361] This experiment investigated the cytotoxicity of anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR8 compared to anti-TPBG-comparative ADC2, which targets 5T4. In MDA-MB-468 cells with high 5T4 levels, anti-TPBG-P5(PEG24)-VC-PAB-exatecan showed a 1.9-fold improvement in cell death IC50 compared to anti-TPBG-comparative ADC2. In BXPC-3 cells with lower 5T4 expression, anti-TPBG-comparative ADC2 showed no activity at all, while anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR8 showed high cytotoxic activity.

[0362] The killing of antigen-positive tumor cells was significantly improved compared to anti-TPBG comparative ADC2. Specificity was demonstrated, as isotype-controlled ADCs did not show efficacy. These results clearly indicate that the conjugate disclosed herein is superior to previously developed ADCs targeting the same target.

[0363] Figure 28 shows the cytotoxic dose-response of anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR 8 and its corresponding non-targeting isotype control conjugate compared to anti-TPBG comparison ADC2 in three different cell lines. The mean and standard deviation of the two measurements, as well as the dose-response fitting, are shown. Fluorescence (in %) compared to the medium control is equal to the percentage of viable cells (%).

[0364] Bystanders die out - the upper layer moves To analyze the bystander activity of ADC against target-negative cells, 20,000 5T4-positive cells (MDA-MB-468 or BXPC-3) were incubated with gradually increasing concentrations of ADC (0-3 μg / ml). After 5 days, half of the cell culture supernatant was transferred to 5,000 5T4-negative cells (SW-620) and incubated for another 5 days. Cell death was analyzed by viability measurement using resazurin as described above.

[0365] In this experiment, the bystander activity of anti-TPBG-P5(PEG24)-VC-PAB-exatecan DAR 8, which targets 5T4, was investigated in a bystander experiment based on supernatant transfer, compared to anti-TPBG comparative ADC2. Anti-TPBG-P5(PEG24)-VC-PAB-exatecan showed high ...

Claims

1. An anti-TPBG antibody (for example, an antibody against TPBG_human trophoblast glycoprotein (e.g., having UniProt accession number: Q13641 or SEQ ID NO: 1)), Anti-TPBG antibodies contain Fc silencing mutations such as leucine (L) to alanine (A) substitutions at positions 234 and 235 (LALA mutations), and (a) Anti-TPBG antibodies are capable of binding to human trophoblast glycoprotein (TPBG) (e.g., those having UniProt accession number: Q13641 or SEQ ID NO: 1); (b) Anti-TPBG antibodies may cross-react with marmoset TPBG (e.g., common marmoset (Callithrix jacchus)) which has white tufts of hair around its ears (e.g., TPBG with UniProtKB accession number: F7I0T3 or SEQ ID NO: 2); and, (c) The anti-TPBG antibody is preferably capable of internal migration via antigen-mediated antibody transfer. Anti-TPBG antibody.

2. The following features: (a) (For example, in MDA-MB-468 cells that endogenously express TPBG (e.g., having DSMZ number: ACC738),) K in the range of about 0.01 to about 10 nmol / L, preferably in the range of about 0.15 to about 0.35 nmol / L, relative to endogenously expressed (e.g., expressed on the cell surface) human TPBG. D Having (More preferably, the K D (This is measured by the FACS assay method.) Most preferably, K in the range of about 0.20 to about 0.30 nmol / L (e.g., 0.25 nmol / L) D Having; and / or (b) Optionally, a K2 solution ranging from about 0.01 to about 10 nmol / L for immobilized exogenous full-length TPBG and / or one or more fragments thereof. D Having (Preferably, the one or more fragments include at least one extracellular domain (ECD) of the TPBG (e.g., the ECD containing at least 32 to 355 amino acids of human TPBG having Uniprot accession number: Q13641 or SEQ ID NO: 1) and / or one or more fragments of the ECD (e.g., having a length of about 15 to about 30 amino acids), and the full-length TPBG and / or one or more fragments thereof are fused to or not fused to one or more protein tags (e.g., 6×His tag, FLAG, HA, V5, Fc fusion, MBP, SUMO, TEV, GFP, TST), and more preferably, the K D The range is approximately 0.05 to approximately 0.30 nmol / L, and most preferably, the K D This is measured by an ELISA assay, and more preferably, the K D This ranges from approximately 0.05 to approximately 0.2 nmol / L (for example, approximately 0.1 to approximately 0.16 nmol / L); (c) Monoclonal antibody; (d) Chimeric antibodies and / or humanized antibodies; (e) Specific recognition of (e.g., human) TPBG that is overexpressed in cancer cells; (f) Human IgG antibody, preferably human IgG1 antibody; (g) Contains kappa (κ) light chain; (h) Contains a lambda (λ) light chain; (i) It is possible for TPBG to be internally transported by target cells expressing TPBG (e.g., cancer cells). (Preferably, the internally transported antibody is directed towards the lysosome); (j) Tumor-selective antibodies (Preferably, the tumor is a liquid tumor and / or a solid tumor, preferably a solid tumor); (k) Antibodies selective for malignant cells; (l) Binding to human TPBG in a glycosylation-dependent manner. (Preferably, the antibody binds to the glycosylated TPBG); and / or (m) Includes Fc silencing mutations such as leucine (L) to alanine (A) substitutions (LALA mutations) at positions 234 and 235. (This LALA mutation can reduce the effector function of immune cells.) An anti-TPBG antibody according to any one of the above claims, comprising one or more of the above.

3. (i) The anti-TPBG antibody has a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 3, and SEQ ID NO: An antibody comprising a light chain variable region having an amino acid sequence that has at least 80% identity with respect to 4 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), Preferably, the anti-TPBG antibody comprises (a) a light chain containing SEQ ID NO: 5 and (b) a heavy chain containing SEQ ID NO: 6; and / or (ii) Anti-TPBG antibody (a') A heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 7, heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 8, and heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 9, (b') Light chain variable region including light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 10, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 11, and light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 12 It is an antibody that contains The anti-TPBG antibody according to any one of the above claims.

4. A hybridoma that produces a monoclonal antibody according to any one of the above claims.

5. An expression vector comprising a nucleic acid encoding an antibody according to any one of the above claims, or at least one of the nucleic acid molecules.

6. An isolated host cell (e.g., an isolated recombinant host cell) comprising the vector and / or nucleic acid according to any one of the claims above.

7. An antibody-drug conjugate (ADC) comprising the anti-TPBG antibody according to any one of the above claims.

8. Anti-TPBG antibody, (a) One or more cytotoxic moieties (e.g., cytotoxic payloads, e.g., tubulin inhibitors, e.g., topoisomerase I inhibitors, e.g., auristatin or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., exatecan) are conjugated, preferably via one or more linkers, more preferably via one or more phosphoamidate linkers; or (b) One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 4 or 8, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) camptothecin (e.g., exatecan) cytotoxic moieties are conjugated preferably via one or more linkers, more preferably via one or more phosphoamidate linkers; (c) One or more exatecan cytotoxic moieties (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) are conjugated via one or more linkers, preferably via one or more phosphoamidate linkers; or (d) One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) camptothecin (e.g., exatecan) cytotoxic moieties are conjugated via conjugation by an ethinylphosphone amidate linker (e.g., to all 8 interchain cysteine ​​residues), Preferably, each phosphone amidate linker has at least one PEG24 portion (for example, to prevent aggregation of the antibody-drug conjugate (ADC)), and more preferably, the ADC has up to eight linker payload portions and eight PEG24 portions. The antibody-drug conjugate (ADC) according to any one of the above claims.

9. (a') The antibody-drug conjugate (ADC) contains a humanized monoclonal TPBG-specific IgG1 antibody conjugated to a cytotoxic payload. (a) The cytotoxic payload is selected from the group consisting of camptothecin, mytansinoids, calicheamycin, duocalmycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof; and / or (b) The cytotoxic payload is a camptothecin moiety C selected from the group consisting of exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, berotecan, lulutotecan, rubitecan, siratecan, cocitecan, and gimatecan; and / or (c) The cytotoxic payload is conjugated via a cleavable linker (L), preferably the linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction, and more preferably the linker is cleavable by a protease, preferably a cathepsin such as cathepsin B; and / or (d) The linker L comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) The cytotoxic payload is an exatecan conjugated via a chemical valine-citrulline-PAB free unit or a valine-alanine-PAB free unit, wherein the free unit is cleavable by a protease and / or (b')(i) An anti-TPBG monoclonal antibody is capable of specifically recognizing (e.g., human) TPBG overexpressed in cancer cells; and (ii) When ADC binds to TPBG overexpressed in cancer cells, the ADC can be internalized by the cell and transported into the lysosomal compartment, where preferably a lysosomal protease (e.g., cathepsin B) can release the cytotoxic payload from the ADC. The antibody-drug conjugate (ADC) according to any one of the above claims.

10. Equation (I): or having a pharmaceutically acceptable salt or solvate thereof, During the ceremony, Ab is the anti-TPBG antibody according to any one of the claims above; is a double bond, or It is a single bond; If it is a double bond, then V does not exist, or When it is a double bond, V is H or (C 1 ~C 8 ) alkyl; If it is a double bond, then X is R 3 -C and or If it is a single bond, then X is and; Y is NR 5 , S, O, or CR 6 R 7 and; R 1 is an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 3 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 4 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 5 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 6 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 7 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; CU stands for connector unit; CM is the cytotoxic part; m is an integer in the range of 1 to 10; and n is an integer in the range of 1 to 20. An antibody-drug conjugate (ADC) according to any one of claims 7 to 9, particularly according to claim 7.

11. is a double bond; V is absent; X is R 3 -C is; and R 3 However, H or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; preferably, R 3 However, H or (C 1 ~C 8 ) alkyl; more preferably, R 3 The antibody-drug conjugate (ADC) according to claim 10, wherein is H.

12. The antibody-drug conjugate (ADC) according to claim 10 or 11, wherein Y is NH.

13. An antibody-drug conjugate (ADC) according to any one of claims 10 to 12, wherein the connector unit CU is detachable.

14. The antibody-drug conjugate (ADC) according to claim 13, wherein the connector unit CU is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction.

15. The antibody-drug conjugate (ADC) according to claim 14, wherein the connector unit CU is cleavable by a protease, preferably a cathepsin, such as cathepsin B.

16. An antibody-drug conjugate (ADC) according to any one of the claims, wherein the connector unit CU comprises a valine-citrulline moiety or a valine-alanine moiety.

17. The connector unit CU is And, In the formula, # indicates a connection point to Y. * This indicates a binding site to the cytotoxic portion CM. The antibody-drug conjugate (ADC) according to claim 16.

18. The connector unit CU is And, During the ceremony, * indicates the binding site to Y, and ## indicates the binding site to the cytotoxic portion CM. The antibody-drug conjugate (ADC) according to claim 16.

19. R 1 but, And, During the ceremony, This indicates the position of O; K F -H, -PO 3 H, -(C 1 ~C 10 )alkyl, -(C 1 ~C 10 )alkyl-SO 3 H, -(C 2 ~C 10 ) Alkyl-CO 2 H, -(C 2 ~C 10 )alkyl-OH, -(C 2 ~C 10 ) Alkyl-NH 2 ,-(C 2 ~C 10 )alkyl-NH(C 1 ~C 3 )alkyl, and -(C 2 ~C 10 )alkyl-N((C 1 ~C 3 )alkyl) 2 Selected from the group consisting of; and o is an integer in the range of 1 to 100. An antibody-drug conjugate (ADC) according to any one of claims 10 to 18.

20. K F The antibody-drug conjugate (ADC) according to claim 19, wherein is H.

21. An antibody-drug conjugate (ADC) according to claim 19 or 20, wherein o is in the range of 8 to 30.

22. The antibody-drug conjugate (ADC) according to claim 21, wherein o is in the range of 20 to 28.

23. The antibody-drug conjugate (ADC) according to claim 22, wherein o is 22, 23, 24, 25, or 26.

24. The antibody-drug conjugate (ADC) according to claim 21, wherein o is in the range of 8 to 16.

25. The antibody-drug conjugate (ADC) according to claim 24, wherein o is 10, 11, 12, 13, or 14.

26. An antibody-drug conjugate (ADC) according to any one of claims 10 to 25, wherein the cytotoxic moiety (CM) is selected from the group consisting of camptothecin, mytansinoids, calicheamycin, duochamycin, tubulisin, amatoxin, drastatin, and auristatin, such as monomethyl auristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof.

27. The antibody-drug conjugate (ADC) according to claim 26, wherein the cytotoxic moiety CM is a camptothecin moiety.

28. The antibody-drug conjugate (ADC) according to claim 27, wherein the camptothecin portion is selected from the group consisting of exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, berotecan, lulutotecan, rubitecan, silatecan, cocitecan, and gimatecan.

29. The cytotoxic part CM is, formula: The antibody-drug conjugate (ADC) according to claim 28, wherein the exatecan has the properties of [the specified exatecan].

30. The cytotoxic part CM is, formula: The antibody-drug conjugate (ADC) according to claim 29, wherein the exatecan has the properties of [the specified property].

31. The antibody-drug conjugate (ADC) according to claim 29 or 30, wherein exatecan is bound to a connector unit CU via an amino group.

32. Ab is the anti-TPBG antibody according to any one of the claims; However, is it a double bond, or However, it is a single bond; If it is a double bond, then V does not exist, or If it is a single bond, then V is H; If it is a double bond, then X is R 3 -C and or If it is a single bond, then X is and; Y is NH; R 1 However, the structure: It is a polyethylene glycol unit having, During the ceremony, This indicates the position of O. K F is H and o is an integer in the range of 8 to 30; R 3 H is; R 4 H is; The CU has the following structure: A connector unit having, where # indicates the connection point to Y, * This indicates the binding site to the camptothecin moiety (CM); CM is the camptothecin portion; m is 1 and n is an integer in the range of 1 to 10. An antibody-drug conjugate (ADC) according to any one of claims 26 to 31.

33. is a double bond; V is absent; X is R 3 -C is; and R 3 The antibody-drug conjugate (ADC) according to claim 32, wherein is H.

34. The camptothecin portion CM is, formula: The antibody-drug conjugate (ADC) according to claim 32 or 33, wherein the exatecan has the properties of exatecan.

35. The antibody-drug conjugate (ADC) according to claim 34, wherein exatecan is bound to a connector unit CU via an amino group.

36. The antibody-drug conjugate (ADC) according to claim 35, wherein o is in the range of 20 to 28.

37. The antibody-drug conjugate (ADC) according to claim 36, wherein o is 22, 23, 24, 25, or 26.

38. An antibody-drug conjugate (ADC) according to any one of claims 32 to 37, wherein n is in the range of 2 to 10, preferably 4 or 8.

39. The following equation (Ia): It has, In the formula, Ab is the anti-TPBG antibody according to any one of the claims above. The antibody-drug conjugate (ADC) according to claim 38.

40. The antibody-drug conjugate (ADC) according to claim 26, wherein the cytotoxic portion CM is auristatin.

41. The antibody-drug conjugate (ADC) according to claim 40, wherein the cytotoxic moiety is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

42. The antibody-drug conjugate (ADC) according to claim 41, wherein the cytotoxic moiety is monomethyl auristatin E (MMAE).

43. The process of conjugating one or more antibodies according to any one of the above claims to one or more cytotoxic moieties (e.g., cytotoxic payloads, e.g., tubulin inhibitors, e.g., topoisomerase I inhibitors, e.g., auristatin or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., exatecan) preferably via one or more linkers, more preferably via one or more phosphoamide linkers. A method for producing antibody-drug conjugates (ADCs) containing [the specified substance].

44. Formula (II) (In the formula, is a triple bond, or It is a double bond; If it is a triple bond, then V does not exist, or If it is a double bond, then V is either H or (C 1 ~C 8 ) is alkyl; If it is a triple bond, then X is R 3 -C and or If it is a double bond, then X is and; Y is NR 5 , S, O, or CR 6 R 7 and; R 1 is an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 3 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 4 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 5 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 6 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 7 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; CU stands for connector unit; CM is the cytotoxic part; and m is an integer in the range of 1 to 10. The compound or a pharmaceutically acceptable salt or solvate thereof Formula (III): (wherein Ab is the anti-TPBG antibody according to any one of the claims above; n is an integer in the range of 1 to 20) molecules containing thiols This is a process of causing a reaction, As a result, equation (I) (In the formula, Ab is the anti-TPBG antibody according to any one of the claims above; In the compound of formula (II) If it is a triple bond, is a double bond, or In the compound of formula (II) If it is a double bond, It is a single bond; If it is a double bond, then V does not exist, or If it is a single bond, then V is either H or (C 1 ~C 8 ) is alkyl; If it is a double bond, then X is R 3 -C and or If it is a single bond, then X is and; Y is NR 5 , S, O, or CR 6 R 7 and; R 1 is an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 3 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 4 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 5 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 6 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; R 7 is H, or an optionally substituted aliphatic residue or an optionally substituted aromatic residue; CU stands for connector unit; CM is the cytotoxic part; m is an integer in the range of 1 to 10; and n is an integer in the range of 1 to 20. A process to obtain an antibody-drug conjugate (ADC), or a pharmaceutically acceptable salt or solvate thereof. The method according to claim 43, including the method described in claim 43.

45. A step of reducing at least one disulfide crosslink of an antibody in the presence of a reducing agent in order to form a thiol group (SH). The method according to claim 44, further comprising:

46. An antibody-drug conjugate (ADC) produced by the method described in any one of the preceding claims.

47. A composition or kit comprising anti-TPBG, antibody-drug conjugate (ADC), hybridoma, nucleic acid, expression vector, and / or host cell according to any one of the above claims, Preferably, the composition is a pharmaceutical and / or diagnostic composition. A composition or kit.

48. A method for treating, improving, preventing, and / or diagnosing cancer, wherein the cancer is selected from the group consisting of breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, cervical cancer, rectal cancer, colon cancer, esophageal cancer, gastric cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcoma, brain cancer, nevus and melanoma, genitourinary cancer, prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancer, retinoblastoma, thyroid cancer, and fallopian tube cancer; more preferably, the cancer is a solid tumor; most preferably, the cancer is breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, uterine cancer (e.g., muscular cancer), uterine cancer. The group consists of: cervical cancer, rectal cancer, colon cancer, anal cancer, esophageal cancer, stomach cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcomas (e.g., osteosarcoma and Kaposi's sarcoma), brain cancer (e.g., pituitary tumors), nevus and melanoma cancer, skin cancer (e.g., squamous cell carcinoma and melanoma), genitourinary cancers (e.g., ureteral and bladder cancer, testicular cancer, prostate cancer, penile cancer), prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancers (e.g., lymphoma, leukemia, myeloma, myelodysplastic syndrome, or myelofibrosis), eye cancers (e.g., retinoblastoma), neuroendocrine tumors, and cancer of unknown primary origin (CUP). The method comprises the step of administering a therapeutically effective or prophylactic amount of the antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit described in any one of the claims, The aforementioned method.

49. (a) as a medicine and / or in a treatment; and / or (b) in the following ways: (i) A method for treating, improving, preventing and / or diagnosing cancer, wherein the cancer is selected from the group consisting of breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, cervical cancer, rectal cancer, colon cancer, esophageal cancer, gastric cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcoma, brain cancer, nevus and melanoma, genitourinary cancer, prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancer, retinoblastoma, thyroid cancer, and fallopian tube cancer; more preferably, the cancer is a solid tumor; most preferably, the cancer is breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, uterine cancer (e.g., muscular cancer), and cervical cancer. A method selected from the group consisting of cancer, rectal cancer, colon cancer, anal cancer, esophageal cancer, stomach cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcoma (e.g., osteosarcoma and Kaposi's sarcoma), brain cancer (e.g., pituitary tumor), nevus and melanoma cancer, skin cancer (e.g., squamous cell carcinoma and melanoma), genitourinary cancer (e.g., ureteral cancer and bladder cancer, testicular cancer, prostate cancer, penile cancer), prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancer (e.g., lymphoma, leukemia, myeloma, myelodysplastic syndrome, or myelofibrosis), eye cancer (e.g., retinoblastoma), neuroendocrine tumors, and cancer of unknown primary origin (CUP); (ii) Methods for monitoring the progression of cancer and / or for evaluating the effectiveness of cancer treatments; (iii) Methods for screening candidate compounds for anticancer activity; (iv) Methods for altering the resistance of cancer cells to chemotherapy; (v) Methods for making cancer cells sensitive to chemotherapy; (vi) Methods for inhibiting the proliferation of cancer cells expressing TPBG; (vii) Methods for the production or preparation of antibodies; (viii) Methods for immunizing non-human animals; (ix) Methods for preparing hybridomas; (x) The method according to any one of the claims above; (xi) any one of (i) to (xi) methods, which is either in vivo, in vitro, or ex vivo. An antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit according to any one of the claims, for use in one or more of the above.

50. below: (a) Treatment, improvement, prevention, and / or diagnosis of cancer (Preferably, the cancer is selected from the group consisting of breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, cervical cancer, rectal cancer, colon cancer, esophageal cancer, stomach cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcoma, brain cancer, nevus and melanoma, genitourinary cancer, prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancer, retinoblastoma, thyroid cancer, and fallopian tube cancer; more preferably, the cancer is a solid tumor; most preferably, the cancer is breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, uterine cancer (e.g., muscular cancer), cervical cancer, rectal cancer, colon cancer, and anal cancer) The following are selected from the group consisting of esophageal cancer, stomach cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, liver cancer, sarcomas (e.g., osteosarcoma and Kaposi's sarcoma), brain cancer (e.g., pituitary tumors), nevus and melanoma cancer, skin cancer (e.g., squamous cell carcinoma and melanoma), genitourinary cancers (e.g., ureteral and bladder cancer, testicular cancer, prostate cancer, penile cancer), prostate cancer, vulvar squamous cell carcinoma, oropharyngeal cancer, endocrine cancer, chest cancer, mesothelioma, pancreatic cancer, bile duct cancer, hematological cancers (e.g., lymphoma, leukemia, myeloma, myelodysplastic syndrome, or myelofibrosis), eye cancers (e.g., retinoblastoma), neuroendocrine tumors, and cancer of unknown primary origin (CUP). (b) Monitoring the progression of cancer and / or evaluating the effectiveness of cancer treatments; (c) Screening candidate compounds for their anticancer activity; (d) Altering the resistance of cancer cells to chemotherapy; (e) Making cancer cells sensitive to chemotherapy; (f) Inhibiting the proliferation of cancer cells that express TPBG; (g) Production or preparation of antibodies; (h) Immunizing non-human animals; (i) Preparation of hybridomas; (j) in the method according to any one of the claims; (k) In vivo, in vitro, or ex vivo use of any one of (a) through (j) Use of one or more of the antibodies, antibody-drug conjugates (ADCs), nucleic acids, expression vectors, host cells, compositions, or kits according to any one of the claims.

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